Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences

ABSTRACT

The present invention is directed to methods and compositions capable of blocking the inhibitory effect of a newly-identified intronic inhibitory sequence element, named ISS-N1 (for “intronic splicing silencer”), located in the SMN2 gene. The compositions and methods of the instant invention include oligonucleotide reagents (e.g., oligoribonucleotides) that effectively target the SMN2 ISS-N1 site in the SMN2 pre-mRNA, thereby modulating the splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. The ISS-N1 blocking agents of the invention cause elevated expression of SMN protein, thus compensating for the loss of SMN protein expression commonly observed in subjects with spinal muscular atrophy (SMA).

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/295,725 filed Dec. 5, 2005, which claims the benefit of U.S. Ser. No.60/633,325, entitled “Spinal Muscular Atrophy (SMA) Treatment ViaTargeting of SMN2 Splice Site Inhibitory Sequences,” filed on Dec. 3,2004. The entire contents of these applications are hereby incorporatedherein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was at least in part provided bythe federal government (N.I.H. grant R01 NS40275). The government may,therefore, have certain rights in the invention.

RELATED INFORMATION

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

BACKGROUND OF THE INVENTION

Alternative splicing increases the coding potential of human genome byproducing multiple proteins from a single gene (Black, D. L. 2003. Annu.Rev. Biochem. 72:291-336). It is also associated with a growing numberof human diseases (Faustino, N. A., and T. A. Cooper. 2003. Genes Dev.17:419-437; Garcia-Blanco, M. A., et al. 2004. Nat. Biotechnol.22:535-546; Pagani, F., and F. E. Baralle. 2004. Nat. Rev. Genet.5:389-396).

Proximal spinal muscular atrophy (SMA) is the second most commonautosomal recessive disorder, and is characterized by the loss of motorneurons in the anterior horn of the spinal cord (Pearn, Lancet 8174,919-922). Linkage mapping identified the Survival of Motor Neuron (SMN)gene as the genetic locus of SMA (Lefebvre et al., Cell 80, 1-5). Inhumans, two nearly identical SMN genes (SMN1 and SMN2) exist onchromosome 5q13. Deletions or mutations within SMN1 but not the SMN2gene cause all forms of proximal SMA (Lefebvre et al., Cell 80, 1-5).SMN1 encodes a ubiquitously expressed 38 kDa SMN protein that isnecessary for snRNP assembly, an essential process for cell survival(Wan, L., et al. 2005. Mol. Cell. Biol. 25:5543-5551). A nearlyidentical copy of the gene, SMN2, fails to compensate for the loss ofSMN1 because of exon 7 skipping, producing an unstable truncatedprotein, SMNΔ7 (Lorson, C. L., et al. 1998. Nat. Genet. 19:63-66). SMN1and SMN2 differ by a critical C to T substitution at position 6 of exon7 (C6U in transcript of SMN2) (Lorson, C. L., et al. 1999. Proc. Natl.Acad. Sci. USA 96:6307-6311; Monani, U. R., et al. 1999. Hum. Mol.Genet. 8:1177-1183). C6U does not change the coding sequence, but issufficient to cause exon 7 skipping in SMN1. Two mutually exclusivemodels have been proposed to explain the inhibitory effect of C6U.According to one model, C6U abrogates an ESE associated with SF2/ASF(Cartegni, L., and A. R. Krainer. 2002. Nat. Genet. 30:377-384), whereasanother model proposes that C6U creates an ESS associated with hnRNP A1(Kashima, T., and J. L. Manley. 2003. Nat. Genet. 34:460-463).

Exon 7 is known to have a weak 3′ ss (Lim, S. R., and K. J. Hertel.2001. J. Biol. Chem. 276:45476-45483), likely due to its suboptimalpolypyrimidine tract. An improved polypyrimidine tract promotedinclusion of exon 7 in SMN2 (Lorson, C. L., and E. J. Androphy. 2000.Hum. Mol. Genet. 9:259-265), indicating that the negative interactionsat C6U and the positive interactions at the polypyrimidine tract weremutually exclusive. Several splicing factors have been implicated inmodulation of SMN exon 7 splicing. Most studied among them has been theSR-like protein, Tra2-β1, that binds to a purine-rich ESE in the middleof exon 7 (Hofmann, Y., et al. 2000. Proc. Natl. Acad. Sci. USA97:9618-23). Elevated expression of Tra2-β1 (ibid.) or its associatedproteins, hnRNP G (Hofmann, Y., and B. Wirth. 2002. Hum. Mol. Genet.11:2037-2049) and Srp30c (Young, P. J., et al. 2002. Hum. Mol. Genet.11:577-587), has been shown to promote exon 7 inclusion in SMN2. Arecent report in which increased expression of STAR (signal transductionand activation of RNA) family of proteins promoted exclusion of exon 7indicated that tissue-specific regulation might occur (Stoss, O., et al.2004. Mol. Cell. Neurosci. 27:8-21). Proteins interacting with intronicsequences could also affect regulation of exon 7 splicing. Consistently,cis-elements present in intron 6 and intron 7 have been shown tomodulate exon 7 splicing (Miyajima, H., et al. 2002. J. Biol. Chem.277:23271-23277; Miyaso, H., et al. 2003. J. Biol. Chem.278:15825-15831). These results have highlighted the complexity ofpre-mRNA splicing, in which exon 7 is defined by a network ofinteractions involving several proteins.

The 54-nucleotide-long exon 7 of human SMN genes contains ˜65% of A+Uresidues. Hence, exon 7 fits into the typical definition of a cassetteexon that generally contains a low percentage of G+C residues (Clark,F., and T. A. Thanaraj. 2002. Hum. Mol. Genet. 11:451-64). In additionto the exon 7 sequence, intronic sequences located immediately upstreamof the 3′ ss or downstream of the 5′ splice site (5′ ss) of SMN2 exon 7have been demonstrated as functionally important in splicing (Miriami,E., et al. 2003. Nucleic Acids Res. 31:1974-1983; Zhang, X. H., and L.A. Chasin. 2004. Genes Dev. 18:1241-1250). These sequences are highlydiverse and can be broadly categorized into G+C-rich and A+U-richregions that constitute distinct pentamer motifs (Zhang, X. H., et al.2005. Genome Res. 15:768-779). Intron 7 sequence downstream of the 5′ ssis rich in A and U residues, but lacks characteristic pentamer motifs.

SMN function correlates with its ability to self-associate (Lorson etal., Nat. Genet. 19, 63-66). SMN also performs a housekeeping role byhelping regenerate the spliceosome through a multi-component SMN complex(Meister et al., Trends Cell Biol. 12, 472-478; Gubitz et al., Exp.Cell. Res. 296, 51-56). Many recent reviews highlight the functionalrole of SMN with direct implications to SMA (Ogino and Wilson, Expert.Rev. Mol. Diagn. 4, 15-29; Iannaccone et al., Curr. Neurol. Neurosci.Rep. 4, 74-80). The defects caused by the lack of SMN1 can be partiallycompensated by high copy number of SMN2, which produces low levels ofthe full-length protein (Monani et al., Hum. Mol. Genet. 9, 2451-2457;Stoilov et al., DNA Cell Biol. 21, 803-818). Most SMA patients have anSMN2 gene, thus, therapies that improve the levels of exon 7 inclusionin SMN2 are likely to be effective.

Antisense technology, used mostly for RNA downregulation, recently hasbeen adapted to alter the splicing process (Kole et al., Acta BiochimPol. (2004) 51, 373-8). Techniques that trick the splicing machinery toalter splicing of SMN2 pre-mRNAs are likely to have high therapeuticvalue.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatantisense targeting, displacement and/or disruption of an intronicsequence in the SMN2 gene can enhance production of full-length SMN2transcripts (transcripts containing exon 7) during splicing. Inparticular, the present inventors have identified a novel intronicinhibitory sequence element, named ISS-N1 (for “intronic splicingsilencer”), in the SMN2 gene as a desirable therapeutic target.Accordingly, the invention is directed to effective use of ISS-N1blocking agents, in particular, blocking oligonucleotide reagents (e.g.,modified antisense oligoribonucleotides) to inhibit this intronicsplice-inhibitory sequence. Treatment of cells derived from SMA patientswith the oligonucleotide reagent compositions of the instant inventioneffectively restored the production of the full-length SMN protein.These results demonstrate for the first time the therapeutic value ofoligonucleotide reagent inhibition of an SMN2 splice site inhibitorydomain, which in exemplary embodiments is achieved through specificinhibition of the ISS-N1 domain using an anti-ISS-N1 (anti-N1)oligonucleotide.

The present invention therefore is directed to compositions capable ofblocking the inhibitory effects of the newly-discovered SMN2 intronicsplice silencing domain, ISS-N1. Agents capable of blocking thesplice-inhibitory effect of this domain have high value as SMAtherapeutics. Featured agents capable of blocking the splice-inhibitoryeffect of the SMN2 ISS-N1 domain include, but are not limited to, e.g.,agents that disrupt the interaction of an ISS-N1-interacting proteinwith the ISS-N1 sequence, agents that sequester an ISS-N1 interactingprotein, agents that disrupt the structure of the ISS-N1 domain and/orsurrounding regions (including, e.g., the U1 snRNP binding site withinthe SMN2 pre-mRNA that lies proximal to the ISS-N1 sequence domain).

In exemplary embodiments, the instant invention is directed tooligonucleotide reagents (e.g., modified antisense oligoribonucleotides)that block the effect on pre-mRNA splicing of the SMN2 ISS-N1 sequencevia direct interaction and/or hybridization with the ISS-N1 sequence.Such RNA-complementary oligonucleotide reagents may be modified byart-recognized means to improve their in vivo stabilities and/orbioaccessibility. The instant invention also is directed to methods foridentifying ISS-N1-interacting proteins, as such methods are enabled bydiscovery and characterization of the ISS-N1 sequence.

In one aspect, the instant invention is directed to an isolatedoligonucleotide reagent (e.g., an oligoribonucleotide) comprising anucleotide sequence which is complementary to an ISS-N1 sequence.

In another aspect, the instant invention is directed to an isolatedoligonucleotide reagent (e.g., an oligoribonucleotide) which iscomplementary to the sequence 5′-CCAGCAUUAUGAAAG-3′ (SEQ ID NO: 3).

In an additional aspect, the instant invention is directed to anisolated oligonucleotide reagent (e.g., an oligoribonucleotide) which iscomplementary to the sequence 5′-CCAGCAUU-3′ (SEQ ID NO: 1).

In a further aspect, the instant invention is directed to an isolatedoligonucleotide reagent (e.g., an oligoribonucleotide) which iscomplementary to the sequence 5′-CCAGCNNNNNGAAAG-3′ (SEQ ID NO: 5).

In another aspect, the instant invention is directed to an isolatedoligonucleotide reagent (e.g., an oligoribonucleotide) which is greaterthan 80% complementary to the sequence 5′-CCAGCAUUAUGAAAG-3′ (SEQ ID NO:3)

In an additional aspect, the instant invention is directed to anisolated oligonucleotide sequence comprising the sequence5′-CUUUCAUAAUGCUGG-3′ (SEQ ID NO: 4).

In another aspect, the instant invention is directed to an isolatedoligonucleotide sequence comprising the sequence 5′-AAUGCUGG-3′ (SEQ IDNO: 2).

In a further aspect, the instant invention is directed to an isolatedoligonucleotide sequence comprising the sequence 5′-CUUUCNNNNNGCUGG-3′(SEQ ID NO: 6).

In another aspect, the instant invention is directed to an isolatedoligonucleotide reagent comprising a sequence greater than 80% identicalto the sequence 5′-CUUUCAUAAUGCUGG-3′ (SEQ ID NO: 4).

In one embodiment, the oligonucleotide is modified by the substitutionof at least one nucleotide with a modified nucleotide, such that in vivostability is enhanced as compared to a corresponding unmodifiedoligonucleotide. In a related embodiment, the modified nucleotide is asugar-modified nucleotide. In another embodiment, the modifiednucleotide is a nucleobase-modified nucleotide.

In an additional embodiment, the modified nucleotide is a 2′-deoxyribonucleotide. In certain embodiments, the 2′-deoxy ribonucleotide is2′-deoxy adenosine or 2′-deoxy guanosine. In another embodiment, themodified nucleotide is a 2′-O-methyl (e.g., 2′-O-methylcytidine,2′-O-methylpseudouridine, 2′-O-methylguanosine, 2′-O-methyluridine,2′-O-methyladenosine, 2′-O-methyl) ribonucleotide. In an additionalembodiment, the modified nucleotide is selected from the groupconsisting of a 2′-fluoro, 2′-amino and 2′-thio modified ribonucleotide.In a further embodiment, the modified nucleotide is selected from thegroup consisting of 2′-fluoro-cytidine, 2′-fluoro-uridine,2′-fluoro-adenosine, 2′-fluoro-guanosine, 2′-amino-cytidine,2′-amino-uridine, 2′-amino-adenosine, 2′-amino-guanosine and2′-amino-butyryl-pyrene-uridine. In an additional embodiment, themodified nucleotide is selected from the group consisting of5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine,2-aminopurine, 5-fluoro-cytidine, and 5-fluoro-uridine,2,6-diaminopurine, 4-thio-uridine, and 5-amino-allyl-uridine.

In a further embodiment, the modified nucleotide is a backbone-modifiednucleotide. In one embodiment, the backbone-modified nucleotide containsa phosphorothioate group. In another embodiment, the modified nucleotideis a locked nucleic acid (LNA).

Another embodiment is directed to a composition comprising anoligonucleotide of the invention. In certain embodiments, thecomposition further comprises a pharmaceutical carrier.

An additional embodiment of the invention is directed to a method ofenhancing the level of exon 7-containing SMN2 mRNA relative toexon-deleted SMN2 mRNA in a cell or cell extract, comprising contactingthe cell or cell extract with an oligonucleotide (e.g., anoligoribonucleotide) of the invention, such that the level of exon7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell orcell extract is enhanced. In one embodiment, the cell or cell extract isa spinal muscular atrophy (SMA) patient-derived neuronal cell, musclecell or fibroblast, or extract thereof. In certain embodiments, the cellor cell extract is selected from the group consisting of an embryonicstem cell, an embryonic stem cell extract, a neuronal stem cell and aneuronal stem cell extract.

A related embodiment of the invention is directed to a method ofenhancing the level of exon 7-containing SMN2 mRNA relative toexon-deleted SMN2 mRNA in an organism, comprising administering to theorganism an oligonucleotide of the invention (e.g., anoligoribonucleotide), such that the level of exon 7-containing SMN2 mRNArelative to exon-deleted SMN2 mRNA in the organism is enhanced. In oneembodiment, the organism is a mammal. In another embodiment, theorganism is a human. In certain embodiments, the human has spinalmuscular atrophy (SMA).

Another embodiment of the invention is directed to a method of treatingspinal muscular atrophy (SMA) in a patient, comprising administering tothe patient an oligonucleotide of the invention (e.g., anoligoribonucleotide) in a dose effective to enhance the level of exon7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in cells ofthe patient, such that SMA in the patient is treated.

A further embodiment is directed to a method for inhibiting an SMN2pre-mRNA intronic splicing silencer site in a cell or cell extractcomprising contacting the cell with an oligonucleotide of the invention(e.g., an oligoribonucleotide), such that the SMN2 intronic splicingsilencer site is inhibited. In a related embodiment, the instantinvention is directed to a method for inhibiting an SMN2 pre-mRNAintronic splicing silencer site in an organism comprising administeringto the organism an oligonucleotide of the invention, such that the SMN2intronic splicing silencer site is inhibited. Another embodiment isdirected to a method for inhibiting an SMN2 pre-mRNA intronic splicingsilencer site in a subject with SMA comprising administering to thesubject an oligonucleotide of the invention (e.g., anoligoribonucleotide), such that the SMN2 intronic splicing silencer siteis inhibited.

An additional aspect of the invention is directed to a method foridentifying a protein that interacts with the ISS-N1 sequence set forthas SEQ ID NO: 1, comprising contacting a cell or cell extract with theISS-N1 sequence under conditions sufficient for the sequence to interactwith a protein in the cell or cell extract; and isolating the ISS-N1sequence and interacting protein, such that the protein that interactswith the ISS-N1 sequence is identified. In one embodiment, the methodfurther comprises UV-crosslinking the ISS-N1 sequence to the interactingprotein. In an additional embodiment, the cell or cell extract is ofmammalian origin. In certain embodiments, the cell or cell extract is ofhuman origin.

Another aspect of the invention is directed to a method of enhancing thelevel of exon 7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNAin a cell or cell extract, comprising contacting the cell or cellextract with an ISS-N1 blocking agent, such that the level of exon7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in the cell orcell extract is enhanced. A related aspect of the invention is directedto a method of enhancing the level of exon 7-containing SMN2 mRNArelative to exon-deleted SMN2 mRNA in an organism, comprising contactingthe organism with an ISS-N1 blocking agent, such that the level of exon7-containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in theorganism is enhanced.

In one embodiment, the ISS-N1 blocking agent is selected from the groupconsisting of a small molecule, a peptide, a polynucleotide, an antibodyor biologically active portion thereof, a peptidomimetic, and anon-peptide oligomer. In an additional embodiment, the ISS-N1 blockingagent is a small molecule.

In another aspect, the invention is directed to a method for inhibitingthe splicing of an exon, comprising insertion of an ISS-N1 sequence at asite within 40 nucleotides of the 5′ splice site of the exon.

In an additional aspect, the invention is directed to a method oftreating amyotrophic lateral sclerosis (ALS) in a patient, comprisingadministering to the patient the oligonucleotide of any of claims 1-21in a dose effective to enhance the level of exon 7-containing SMN2 mRNArelative to exon-deleted SMN2 mRNA in cells of the patient.

In an additional embodiment, the oligonucleotide reagent of theinvention is a ribozyme.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a model of cis-elements that regulate splicing of exon 7of human SMN, wherein upper-case letters represent exon 7 sequences,lower-case letters represent intronic sequences, and the asterisk (*)represents position 6, where C is replaced by U (C6U) in SMN2 exon 7(SEQ ID NO: 67). The U1 snRNA binding site that spans the first sixnucleotides of intron 7 is also highlighted.

FIGS. 2A and B show the effect on SMN2 splicing of mutations in SMN2intron 7, some of which restored strong U1 snRNA base-pairing at the 5′splice site (ss) of intron 7, thus identifying the ISS-N1 sequencedomain. FIG. 2A depicts positions of the mutations (SEQ ID NOS 68-70).FIG. 2B shows the splicing patterns of SMN2 mutants, with Tra2-ESE(−)mutants harboring abrogated Tra2-ESE, performed similarly as in Singh etal., RNA 10, 1291-1305.

FIGS. 3A and B depict the randomization and selection approach used toanalyze the eight-nucleotide-long intronic cis-element ISS-N1 (whichlies just after the U1 snRNA binding site at the 5′ ss of intron 7).FIG. 3A schematically depicts the eight-nucleotide-long sequence (SEQ IDNO: 71) stretch in which six nucleotide positions (marked as “N”) wererandomized to generate the initial pool of mutants tested for alterationof SMN2 splicing. Sequences of a randomly-picked eleven clones (from theinitial pool) are shown. FIG. 3B shows the SMN2 splicing pattern ofthese randomly-picked eleven clones, with mutant 033 demonstrated tofully restore exon 7 inclusion in the processed SMN2 transcript.

FIGS. 4A-C show the effect of intronic deletions downstream of the 5′ ssof exon 7 of SMN2. FIG. 4A depicts intronic sequences of SMN2 deletionmutants (SEQ ID NOS 72-87, respectively in order of appearance).Nucleotide numbering starts from the beginning of intron 7, anddeletions are shown as dashed lines. The ISS-N1 site is shaded.Nucleotides involved in base pairing with U1 snRNA are shown in bold andhighlighted. Numbers in mutants' names represent the span of positionsdeleted. FIG. 4B shows the in vivo splicing pattern of the SMN2 deletionmutants shown in panel A. The upper band corresponds to fully-splicedproduct that includes exon 7; the lower band corresponds to exon7-skipped product. The percent of exon 7 skipping was calculated fromthe total value of exon-7-included and exon-7-skipped products.Abbreviations E6, E7, and E8 stand for exon 6, exon 7, and exon 8,respectively. FIG. 4C shows the in vivo splicing pattern of SMN2ΔISS-N1(ISS-N1-deleted mutant) in different cell lines. Notably, SMN2ΔISS-N1was the same construct as the N1Δ10-24 mutant in FIG. 4A. Cell linesused were Neuro-2a (mouse brain neuroblastoma, lanes 1-3), NSC34 (mousemotor neuron-like, lanes 4-6), SK-N-SH (human neuroblastoma, lanes 7-9),P-19 (mouse embryonal teratocarcinoma, lanes 10-12), HEK293 (humanembryonal kidney cells, lanes 13-15). Spliced products were the same asindicated in FIG. 4B.

FIGS. 5A and B display the evolutionary significance of ISS-N1. FIG. 5Ashows an alignment of the first 42 nucleotides of human and mouse intron7, including the ISS-N1 region. Sequence numbering starts from thebeginning of intron 7, with the ISS-N1 sequence shaded. Nucleotidesinvolved in base pairing with U1 snRNA are shown in bold andhighlighted. In the top two lines, homologous positions between humanand mouse sequences are shaded in black. In the bottom ten lines,intronic sequences of SMN2 mutants (SMN2/MS1 through SMN2/MS10) areshown with mouse nucleotides written in lower-case letters andhighlighted in black (SEQ ID NOS 88-99, respectively in order ofappearance). FIG. 5B shows the in vivo splicing pattern of SMN2 mutantsshown in FIG. 5A, with spliced products the same as indicated in FIG.4B.

FIG. 6 schematically depicts the RNA structure of wild-type (SEQ ID NO:101), mutant 033 (SEQ ID NO: 102) and mutant A54G (SEQ ID NO: 100)pre-mRNAs. Numbering starts from exon 7. Exon 7 sequence is shown inupper-case letters, while intron 7 sequences are shown in lower-caseletters. The stem loop structure of TSL2 is shaded dark, whilelight-shaded regions are regions of co-axial stacking.

FIGS. 7A-C depict the effect of treatment with an antisenseoligonucleotide (anti-N1) on splicing of exon 7 in SMN2. FIG. 7A depictsthe sequence of the anti-N1 oligonucleotide (SEQ ID NO: 39) and itsannealing site within intron 7 of the SMN2 pre-mRNA (SEQ ID NO: 103).FIG. 7B shows that the anti-N1 oligonucleotide mediated restoration ofexon 7 inclusion in SMN2 when transiently transfected to cells. Theprominent antisense effect was observed even at the lowest concentrationof the anti-N1 oligonucleotide (25 nM). FIG. 7C shows that the anti-N1oligonucleotide had no effect on splicing of mutants that have a deletedor mutated ISS-N1 site. In ΔN1S and ΔN1L mutants, ISS-N1 was completelydeleted, while in Mut-N1, the ISS-N1 domain is mutated. Lack ofimprovement of exon 7 inclusion in the presence of anti-N1oligonucleotide confirmed that the effect of anti-N1 oligonucleotide wassequence-specific.

FIGS. 8A-C show the effect of antisense oligonucleotides on splicing ofdifferent minigenes. FIG. 8A (SEQ ID NO: 104) depicts a diagrammaticrepresentation of intron 7 regions targeted by antisenseoligonucleotides Anti-N1, Anti-N1+10, Anti-N1+20 and Anti-N1+30.Sequence numbering starts from the beginning of intron 7. The ISS-N1region is highlighted. Of the four oligonucleotides shown, only Anti-N1fully sequestered ISS-N1. FIG. 8B shows the in vivo splicing pattern ofSMN2 minigene in the presence of antisense oligonucleotides. Splicedproducts are the same as indicated in FIG. 4B. FIG. 8C shows the in vivosplicing pattern of different minigenes in the presence of Anti-N1. Inevery lane, the upper band represents the exon included, whereas thelower band represents the exon-excluded products. For CFTR, apoA-II, Fasand Casp3, the major bands represent the exon-included products. For Tauand Fas (mut), the major bands represent the exon-excluded products. Fordetection of spliced products in lanes 1-6, 9 and 10, cells wereharvested 40 hours after transfection (for other lanes, refer toMaterials and Methods).

FIGS. 9A-C show the effect of base-pairing between Anti-N1 and ISS-N1 onthe efficiency of SMN2 exon 7 inclusion. FIG. 9A shows the intron 7sequences of SMN2 and mutants with substitutions in the ISS-N1 region(SEQ ID NOS 72 and 105-107, respectively in order of appearance).Sequence numbering starts from the beginning of intron 7. The ISS-N1region is highlighted in grey. Nucleotides involved in base pairing withU1 snRNA are shown in bold and highlighted. Note that intronic mutations(highlighted in black) abrogate base pairing between Anti-N1 and ISS-N1.FIG. 9B shows the nucleotide sequence of Anti-N1 (SEQ ID NO: 39) andAnti-I7-25 (SEQ ID NO: 108) oligonucleotides. Differences arehighlighted in black. Note that Anti-I7-25 will restore base pairingwith ISS-N1 region in mutant SMN2/I7-25. FIG. 9C shows the in vivosplicing pattern of mutants shown in FIG. 9A. Plasmid DNA (0.1 μg) wastransfected alone or co-transfected with 50 nM of Anti-N1 or Anti-I7-25oligonucleotide. Spliced products were the same as indicated in FIG. 4B.

FIGS. 10A and B show the relative significance of exon 7 cis-elements ascompared to ISS-N1. FIG. 10A depicts a diagrammatic representation ofseveral cis-elements involved in regulation of exon 7 splicing (not tothe scale). Elements 1G, Tra2-β1 (Tra2-ESE), CT (Conserved Tract) andelement 2 represent positive elements (marked as “+”). The ISS-N1 domainis a negative element (marked as “−”). FIG. 10B shows the in vivosplicing pattern of SMN1 mutants, in which deletion of ISS-N1 (combinedwith abrogation of a given positive cis-element. Spliced products werethe same as indicated in FIG. 4B. Abr-E2 represents abrogation ofelement 2 by a triple substitution G69C/U70A/U71A as in intron 7(Miyaso, H., et al. 2003. J. Biol. Chem. 278:15825-15831), Abr-Tra2represents abrogation of Tra2-ESE by 25U26U mutation in exon 7 (Hofmann,Y., et al. 2000. Proc. Natl. Acad. Sci. USA 97:9618-23), 1U mutationrepresents abrogation of a cis-element at the first position (Singh, N.N., et al. 2004. RNA 10:1291-1305) and Abr-CT represents abrogation ofconserved tract by 36U37U mutation in exon 7 (ibid.).

FIGS. 11A-D demonstrate the portability of the ISS-N1 element. In FIG11A, the upper panel shows the location of ISS-N1 within SMN2 intron 7with respect to the 5′ ss (SEQ ID NOS 109-113, respectively in order ofappearance). The ISS-N1 sequence was inserted at different locationswithin intron 7 of SMN2SMN2 mutants with 5-nucleotide-long insertionsimmediately upstream of ISS-N1 (SEQ ID NOS 72 and 114-117, respectivelyin order of appearance). Nucleotide position and types of insertions areindicated. Sequence numbering starts from the beginning of intron 7. TheISS-N1 sequence is shaded. Nucleotides involved in base pairing with U1snRNA are shown in bold and highlighted. FIG 11B shows the in vivosplicing pattern of mutants shown in FIG. 11A. Spliced products were thesame as indicated in FIG. 4B. FIG. 11C shows the effect of insertion ofISS-N1 in a heterologous context. For insertion of the ISS-N1 sequence,Avr II restriction site was first inserted downstream of exon 6 of Casp3minigene (SEQ ID NOS 118 and 119). FIG. 11D shows the in vivo splicingpattern of mutants shown in FIG. 11C. The splicing pattern wasdetermined in the absence and presence of antisense oligonucleotide(Anti-ISS-N1/15) that fully sequestered ISS-N1. In the absence ofAnti-ISS-N1/15, Casp3ISS-N1 mutant increased exclusion of Casp3 exon 6(compare lane 3 with 4).

FIGS. 12A-D show the effect of Anti-N1 treatment on the splicing ofendogenous genes. FIG. 12A shows the splicing of endogenous SMN2 afterSMA fibroblasts (GM03813) were transfected with 5 nM ofoligonucleotides. Total RNA was collected 25 hours after transfection.FIG. 12B demonstrates the specificity of Anti-N1 on the splicing patternof other exons in SMA fibroblasts (GM03813). The sizes of the expectedspliced products are indicated to the left. The same RNA used in FIG.12A (lanes 2 and 3) was used for this analysis. The 662-bp band in lanes1 and 2 represents SMN-exon-3-included product, whereas 387-bp band inlanes 3 and 4 represent SMN-exon-5-included product. The 440-bp band inlanes 5 and 6 represents transcripts that exclude exons 2b but includeexon 3 of Survivin (Mahotka, C., et al. 1999. Cancer Res. 59:6097-6102).The 830 by band in lanes 7 and 8 represents transcripts that includeexons 29 and 30 of NF1 (Park, V. M., et al. 1998. Hum. Genet.103:382-385). The 686 by band in lanes 9 and 10 represents transcriptsthat produce Tra2-β1 spliced variant of Tra2 (Chen, X., et al. 2003.Cell Biol. Int. 27:491-496). The 474 by band in lanes 11 and 12represents Caspase 3 exon 6 included product (Huang, Y., et al. 2001.Biochem. Biophys. Res. Commun. 283:762-769). The 300 by band in lanes 13and 14 represents Bcl-xL spliced variant of Bcl-x (Mercatante, D. R., etal. 2002. J. Biol. Chem. 277:49374-49382). FIG. 12C shows antisenseoligonucleotide-mediated (anti-N1-mediated) restoration of SMN proteinin patient cells. The antisense effect was verified at the protein levelfor SMA patient-derived fibroblasts. SMA fibroblasts were transfectedwith anti-N1 oligonucleotide and cell lysates were prepared 48 and 72hours post-transfection. As shown in lanes 1 and 4, the level of SMNprotein increased as compared to the untransfected cells (lanes 2 and5). To ensure even protein loading, membranes were stained with SyproRuby Protein Blot™ stain (Bio-Rad). In addition, SMN protein levels werecompared to alpha-tubulin levels as an internal control (though elevatedalpha-tubulin levels have consistently been observed in patientfibroblasts as compared to normal fibroblasts). FIG. 12D also shows theeffect of Anti-N1 on the level of SMN protein. Western blots wereperformed to detect SMN in SMA fibroblasts (GM03813) transfected with 5nM (lane 1) and 15 nM (lane 3) of Anti-N1. GM03813 cells transfectedwith control oligonucleotide Scramble20 (lanes 2 and 4), and mocktransfected GM03813 cells (lane 5) or AG06814 cells (normal fibroblasts)(lane 6) were used as controls. For detection of SMN, cells wereharvested 72 hours after transfection. α-tubulin was used as a loadingcontrol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that targeting of anintronic sequence in the SMN2 gene can enhance production of full-lengthSMN2 transcripts (transcripts containing exon 7) during splicing. Inparticular, the present inventors have identified a novel intronicinhibitory sequence element, named ISS-N1 (for “intronic splicingsilencer”), in the SMN2 gene as a valuable therapeutic target.Accordingly, the invention is directed to effective use of blockingcompounds, in particular, oligonucleotide reagents (e.g., modifiedantisense oligoribonucleotides) to inhibit this intronicsplice-inhibitory sequence. The ISS-N1 sequence motif was identified toplay a dominant role in production of exon 7-deleted SMN2 transcripts.Oligoribonucleotide reagents complementary to ISS-N1 were shown toenhance inclusion of exon 7 during splicing of SMN2 transcript in SMApatient fibroblasts, thus restoring production of full-length SMN2 mRNAtranscripts. Consequently, this treatment also restored the productionof the full-length SMN protein. Displacement and/or disruption of theISS-N1 site was also shown to restore inclusion of exon 7 in SMNtranscripts; thus, the invention is also directed to therapies thatdisplace and/or disrupt the ISS-N1 sequence. These results demonstratedfor the first time the therapeutic value of inhibition of SMN2 splicesite inhibitory domains, for example, through specific inhibition of theISS-N1 domain using an anti-ISS-N1 (anti-N1) oligonucleotide.

The present invention provides compositions for blocking the inhibitoryeffects of the newly-discovered SMN2 intronic splice silencing domain,ISS-N1. In particular, the invention provides compositions comprisingoligonucleotide reagents (e.g., antisense agents or dsDNA cassettes)that block the splice inhibitory effects of the ISS-N1 domain, therebymodulating splicing of the SMN2 pre-mRNA to include exon 7 in processedforms of the transcript. Agents capable of blocking the splicing effectof ISS-N1 have high value as SMA therapeutics. Such agents can also beused in treatment of amyotrophic lateral sclerosis (ALS), anotherneurological disease characterized by low levels of SMN protein(Veldink, J. H., et al. 2005 Neurology 65(6):820-5). The inventiontherefore provides agents capable of blocking the splice-inhibitoryeffect of the SMN2 ISS-N1 domain, including but not limited to, e.g.,agents that disrupt the interaction of an ISS-N1-interacting proteinwith the ISS-N1 sequence, agents that sequester an ISS-N1 interactingprotein, agents that disrupt the structure of the ISS-N1 domain and/orsurrounding regions (including, e.g., the U1 snRNP binding site withinthe SMN2 pre-mRNA that lies proximal to the ISS-N1 sequence domain).

In exemplary embodiments, the instant invention is directed tooligonucleotide reagents capable of blocking the effect on pre-mRNAsplicing of the SMN2 ISS-N1 sequence via direct interaction and/orhybridization. To enhance the therapeutic value of suchRNA-complementary oligonucleotides, the invention is further directed tocompositions comprising modified forms of such oligonucleotides, e.g.,phosphorothioate-, 2′-O-methyl-, etc.-modified oligonucleotides, as suchmodifications have been recognized in the art as improving the stabilityof oligonucleotides in vivo. The instant invention also is directed tomethods for identifying ISS-N1-interacting proteins, as such methods areenabled by the instant discovery and characterization of the ISS-N1sequence.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. The term “nucleotide” refers to a nucleoside having one ormore phosphate groups joined in ester linkages to the sugar moiety.Exemplary nucleotides include nucleoside monophosphates, diphosphatesand triphosphates. The terms “polynucleotide” and “nucleic acidmolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides. The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Preferred nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivitized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

As used herein, the term “intronic splicing silencer-N1” or “ISS-N1”refers to the sequence 5′-CCAGCAUUAUGAAAG-3′ (SEQ ID NO: 3), or anysequence or variant thereof capable of inhibiting the inclusion of exon7 during splicing of the SMN2 pre-mRNA. One such effective sequencethereof is 5′-CCAGCAUU-3′ (SEQ ID NO: 1). Critical residues that mediatethe splice site inhibitory activity of the ISS-N1 sequence can also berepresented by the sequence 5′-CCAGCNNNNNGAAAG-3′ (SEQ ID NO: 5). Thus,any such sequence that acts to inhibit inclusion of exon 7 duringsplicing of the SMN2 pre-mRNA can be referred to simply as an “ISS-N1sequence.”

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. An “oligonucleotide reagent” of the inventionincludes any agent, compound or composition that contains one or moreoligonucleotides, and includes, e.g., reagents comprising both singlestranded and/or double stranded (ds) oligonucleotide compositions,including, e.g., single stranded RNA, single stranded DNA, DNA/DNA andRNA/DNA hybrid compositions, as well as derivatized/modifiedcompositions thereof. Such “oligonucleotide reagents” may also includeamplified oligonucleotide products, e.g., polymerase chain reaction(PCR) products. An “oligonucleotide reagent” of the invention may alsoinclude art-recognized compositions designed to mimic the activity ofoligonucleotides, such as peptide nucleic acid (PNA) molecules.

The term “oligoribonucleotide” refers to a short polymer ofribonucleotides and/or ribonucleotide analogs.

An “oligoribonucleotide” of the invention can include one or a fewdeoxyribonucleotides or deoxyribonucleotide analogs in order to enhancethe stability and/or bioaccessibility of the molecule, however, thechemical nature of the entire molecule must be primarily of aribonucleotide nature in order that ISS-N1 blocking activity occursabsent degradation of the target RNA (i.e., absent the RNase Hdegradation triggered by oligodeoxyribonucleotides or DNA:RNAhybridization).

Preferably, the oligonucleotide reagent molecules/agents of theinvention act (or are effective) at a concentration (e.g., have an IC50)in the nanomolar range, for example, less than 500 nM, preferably lessthan 400 nM, more preferably less than 300, 250, 200, 150, 100, 75, 50,25, 10, 5, 2 or 1 nM.

Preferred oligonucleotide reagent molecules/agents are modifiedoligonucleotides having a length of about 5 to 50 nucleotides (ornucleotide analogs), e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50nucleotides (or nucleotide analogs). In preferred embodiments,oligonucleotide reagent molecules/agents are modified oligonucleotideshaving a length of about 15 to 40 nucleotides (or nucleotide analogs).In other embodiments, oligonucleotide reagent molecules/agents aremodified oligonucleotides having a length of about 3 to 80 nucleotides(or nucleotide analogs), or for example, about 3-10, 10-15, 15-20,20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70,70-75, 75-80 or 80 or more nucleotides (or nucleotide analogs).

The term “agent” and “compound” are used interchangeably herein.

As used herein, the term “nuclease-resistant oligonucleotide” refers toany oligonucleotide that has been modified to inhibit degradation byenzymes such as, for example, the exonucleases known to be present inthe cytoplasm of a eukaryotic cell. RNA molecules (e.g., RNAoligonucleotides) are particularly at risk of degradation when combinedwith a composition comprising a cell extract or when introduced to acell or organism, and a “ribonuclease-resistant” oligonucleotide is thusdefined as an oligonucleotide reagent molecule/agent that is relativelyresistant to ribonuclease enzymes (e.g., exonucleases), as compared toan unmodified form of the same oligonucleotide. Preferredoligonucleotide reagent molecules/agents of the invention include thosethat have been modified to render the oligonucleotide relativelynuclease-resistant or ribonuclease-resistant. In a preferred embodiment,the oligonucleotide reagents of the invention have been modified with a2′-O-methyl group (e.g., 2′-O-methylcytidine, 2′-O-methylpseudouridine,2′-O-methylguanosine, 2′-O-methyluridine, 2′-O-methyladenosine,2′-O-methyl) and additionally comprise a phosphorothioate backbone.

The terms “2′-O-methyl modification”, “phosphorothioate modification”and “locked nucleic acid” (LNA; oligonucleotides comprising at least one2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer), as usedherein, possess their art-recognized meanings.

The term “antisense” refers generally to any approach reliant uponagents, e.g., single-stranded oligonucleotides, that are sufficientlycomplementary to a target sequence to associate with the target sequencein a sequence-specific manner (e.g., hybridize to the target sequence).Exemplary uses of antisense in the instant application involve use of anoligoribonucleotide agent that hybridizes to a target pre-mRNA moleculeand blocks an activity/effect (e.g., splicing pattern) of the targetedpre-mRNA sequence, but antisense approaches commonly are used to targetDNA or RNA for transcriptional inhibition, translational inhibition,degradation, etc. Antisense is a technology that can be initiated by thehand of man, for example, to modulate splicing and/or silence theexpression of target genes.

As used herein, the term “antisense oligonucleotide” refers to a nucleicacid (in preferred embodiments, an RNA) (or analog thereof), havingsufficient sequence complementarity to a target RNA (i.e., the RNA forwhich splice site selection is modulated) to block a region of a targetRNA (e.g., pre-mRNA) in an effective manner. In exemplary embodiments ofthe instant invention, such blocking of the ISS-N1 domain in SMN2pre-mRNA serves to modulate splicing, either by masking a binding sitefor a native protein that would otherwise modulate splicing and/or byaltering the structure of the targeted RNA. In preferred embodiments ofthe instant invention, the target RNA is a target pre-mRNA (e.g., SMN2pre-mRNA). An antisense oligonucleotide having a “sequence sufficientlycomplementary to a target RNA sequence to modulate splicing of thetarget RNA” means that the antisense agent has a sequence sufficient totrigger the masking of a binding site for a native protein that wouldotherwise modulate splicing and/or alters the three-dimensionalstructure of the targeted RNA Likewise, an oligonucleotide reagenthaving a “sequence sufficiently complementary to a target RNA sequenceto modulate splicing of the target RNA” means that the oligonucleotidereagent has a sequence sufficient to trigger the masking of a bindingsite for a native protein that would otherwise modulate splicing and/oralters the three-dimensional structure of the targeted RNA

As used herein, the terms “ISS-N1 blocking agent,” “ISS-N1 blocker,” and“ISS-N1 blocking compound” refer to any agent (e.g., oligonucleotide,oligoribonucleotide, small molecule, etc.) that is capable of inhibitingthe effect of the SMN2 ISS-N1 site (e.g., lessen the inhibition of SMN2exon 7 inclusion during splicing that is caused by the ISS-N1 site).

As used herein, the term “antisense strand” as it pertains to anoligonucleotide reagent refers to a strand that is substantiallycomplementary to a section of about 10-50 nucleotides, e.g., about15-30, 16-25, 18-23 or 19-22 nucleotides of the pre-mRNA targeted formodulation of splicing. The antisense strand has sequence sufficientlycomplementary to the desired target pre-mRNA sequence to directtarget-specific modulation of RNA splicing (e.g., complementaritysufficient to trigger the formation of a desired target mRNA throughmodulation of splicing via, e.g., altered recruitment of the splicingmachinery or process).

As used herein, the “5′ end”, as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end”, as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

An oligonucleotide reagent “that directs altered RNA splicing of a gene”is an oligonucleotide that has a sequence sufficiently complementary tothe target mRNA encoded by a gene to trigger altered splicing of thetarget mRNA by the splicing machinery or process, or, alternatively, isan oligonucleotide reagent that displaces and/or disrupts the sequenceof ISS-N1.

As used herein, the term “isolated sequence” (e.g., “isolatedoligonucleotide” or “isolated oligoribonucleotide”) refers to sequenceswhich are substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

A “target gene” is a gene whose splicing is to be selectively modulated.This modulation is achieved by altering the splicing pattern of thepre-mRNA of the target gene (also referred to herein as the “targetpre-mRNA”) with an oligonucleotide reagent, resulting in an alteredprocessed mRNA (the “target mRNA”). In certain embodiments, theoligonucleotide reagent is complementary, e.g., sufficientlycomplementary to, e.g., a section of about 18 to about 40 or morenucleotides of the pre-mRNA of the target gene to trigger the alteredsplicing of the pre-mRNA of the target gene. Alternatively, theoligonucleotide reagent is sufficiently homologous to the ISS-N1sequence and/or sequences surrounding the ISS-N1 sequence to causedisruption and/or displacement of the ISS-N1 sequence from the 5′ ss ofa targeted exon (in preferred embodiments, SMN2 exon 7) uponsequence-specific integration (e.g., homologous recombination) at suchsequences.

As used herein, the term “SMA” refers to spinal muscular atrophy, ahuman autosomal recessive disease that is often characterized byunderexpression of SMN protein in affected individuals.

As used herein the term “compound” includes any reagent which is testedusing the assays of the invention to determine whether it modulatessplice site modulation, e.g., oligonucleotide reagent-mediated splicingmodulation. More than one compound, e.g., a plurality of compounds, canbe tested at the same time for their ability to modulate splicing in ascreening assay.

In one embodiment, test compounds comprise any selection of the groupconsisting of a small molecule (e.g., an organic molecule having amolecular weight of about 1000 Da or less), a peptide, a polynucleotide,an antibody or biologically active portion thereof, a peptidomimetic,and a non-pepdide oligomer.

A gene “involved” in a disorder includes a gene, the normal or aberrantexpression or function of which effects or causes a disease or disorderor at least one symptom of said disease or disorder.

As used herein, the term “ribozyme” refers to a nucleic acid moleculewhich is capable of cleaving a specific nucleic acid sequence. Ribozymesmay be composed of RNA, DNA, nucleic acid analogues (e.g.,phosphorothioates), or any combination of these (e.g., DNA/RNAchimerics). Within certain embodiments, a ribozyme should be understoodto refer to RNA molecules that contain antisense sequences for specificrecognition, and an RNA-cleaving enzymatic activity.

Various methodologies of the invention include a step that involvescomparing a value, level, feature, characteristic, property, etc. to a“suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an oligonucleotide reagentmethodology, as described herein. For example, a transcription rate,mRNA level and/or splicing pattern, translation rate, protein level,biological activity, cellular characteristic or property, genotype,phenotype, etc. can be determined prior to introducing anoligonucleotide reagent (e.g., an oligonucleotide, compound, etc., thatalters splicing of target pre-mRNA in a sequence-specific manner) of theinvention into a cell or organism. In another embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined in a cell or organism, e.g., acontrol or normal cell or organism, exhibiting, for example, normaltraits. In yet another embodiment, a “suitable control” or “appropriatecontrol” is a predefined value, level, feature, characteristic,property, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Oligonucleotide Reagents and Splice Site Alteration

The present invention is directed to oligonucleotide reagents, e.g.,antisense oligonucleotides, suitable for use in blocking a domain of atarget RNA (in exemplary embodiments, a pre-mRNA is blocked, therebymodulating splice site selection of the mRNA splicing machinery) both invitro and in vivo. In vivo methodologies are useful for both generalsplice site modulatory purposes as well as in therapeutic applicationsin which blocking of a target mRNA domain (e.g., enhancement of splicesite selection via oligonucleotide reagent-mediated inhibition of asplice site inhibitor domain) is desirable. Oligonucleotide reagents ofthe invention are of any size and/or chemical composition sufficient toblock a target RNA (e.g., pre-mRNA), in particular exemplaryembodiments, the reagent is of any size and/or chemical compositionsufficient to inhibit the ISS-N1 intronic splice silencing domain ofSMN2. In exemplary embodiments, the oligonucleotide reagents of theinvention are oligonucleotides of between about 5-300 nucleotides (ormodified nucleotides), preferably between about 10-100 nucleotides (ormodified nucleotides; e.g., ribonucleotides or modifiedribonucleotides), for example, between about 15-35, e.g., about 15-20,20-25, 25-30, 30-35 (31, 32, 33, 34, 35), or 35-40 nucleotides (ormodified nucleotides; e.g., ribonucleotides or modifiedribonucleotides). Oligonucleotide reagents are preferablysufficiently-complementary to target RNA sequences, in particularembodiments, the intronic ISS-N1 domain sequence of the SMN2 pre-mRNA.In exemplary embodiments of the invention, oligonucleotide reagentscomprise oligonucleotides that contain phosphorothioate and 2′-O-methyl(e.g., 2′-O-methylcytidine, 2′-O-methylpseudouridine,2′-O-methylguanosine, 2′-O-methyluridine, 2′-O-methyladenosine,2′-O-methyl) modifications. Many other forms of oligonucleotidemodification may be used to generate oligonucleotide reagents of theinstant invention, including, for example, locked nucleic acids(oligonucleotides comprising at least one 2′-C,4′-C-oxy-methylene-linkedbicyclic ribonucleotide monomer), with one of skill in the artrecognizing other modifications capable of rendering an oligonucleotidereagent effective for inducing inclusion of a target exon during RNAsplicing (especially as relates to in vivo stability of theoligonucleotide reagents—refer to “Modifications” section below).

An oligonucleotide reagent can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45, or 50 or more nucleotides in length. An oligonucleotidereagent of the invention can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an oligonucleotide reagent (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the oligonucleotide reagent can beproduced biologically using an expression vector into which a nucleicacid has been sub-cloned, e.g., in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The oligonucleotide reagents of the invention are typically administeredto a subject or generated in situ such that they hybridize with or bindto cellular pre-mRNA and/or genomic DNA comprising an ISS-N1 sequence tothereby inhibit inclusion of an exon during splicing. The hybridizationcan be by conventional nucleotide complementarity to form a stableduplex, or, for example, in the case of an oligonucleotide reagent whichbinds to DNA duplexes, through specific interactions in the major grooveof the double helix. Examples of a route of administration ofoligonucleotide reagents of the invention include direct injection at atissue site or infusion of the antisense nucleic acid into anappropriately-associated body fluid, e.g., cerebrospinal fluid.Alternatively, oligonucleotide reagents can be modified to targetselected cells and then administered systemically. For example, forsystemic administration, oligonucleotide reagents can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the oligonucleotide reagents topeptides or antibodies which bind to cell surface receptors or antigens.The oligonucleotide reagents can also be delivered to cells using thevectors described herein. To achieve sufficient intracellularconcentrations of the oligonucleotide reagents, vector constructs inwhich the oligonucleotide reagent is placed under the control of astrong pol II or pol III promoter are preferred.

An oligonucleotide reagent of the invention can be an α-anomeric nucleicacid molecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual α-units, the strands run parallel to each other (Gaultier et al.,1987, Nucleic Acids Res. 15:6625-6641). The oligonucleotide reagent canalso comprise a 2′-o-methylribonucleotide (Inoue et al., 1987, NucleicAcids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.,1987, FEBS Lett. 215:327-330).

In various embodiments, the oligonucleotide reagents of the inventioncan be modified at the base moiety, sugar moiety or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacid molecules can be modified to generate peptide nucleic acidmolecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs”refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al. (1996), supra; Perry-O′Keefe et al. (1996)Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O′Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675). In certain embodiments of theinstant invention, PNAs can also be generated to target an ISS-N1sequence.

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNASE H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17): 3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5: 1119-11124).

In certain embodiments of the present invention, a PNA compound thatbinds to an ISS-N1 sequence can be generated additionally to contain oneor more charged groups. Such tethering of charged groups to anti-ISS-N1compounds can improve the delivery and/or activity of the anti-ISS-N1compounds of the invention, or also can be used to minimize non-specificeffects potentially associated with alternative other formulations ofthe oligonucleotide reagents of the instant invention. In oneembodiment, the oligonucleotide reagents of the invention can begenerated as phosphono-PNA molecules (pPNAs), wherein one or morephosphate groups are attached to and/or incorporated into the backboneof the oligonucleotide reagent (refer to Efimov, V., et al.2003Nucleosides, Nucleotides & Nucleic Acids 22(5-8): 593-599,incorporated in its entirety herein by reference).

In further embodiments, the oligonucleotide reagents of the inventioncan be generated as gripNA™ compounds. GripNA™ molecules are a form ofnegatively charged PNA, which exhibit greater sequence specificitycompared to conventional oligonucleotide reagents (e.g., antisense/genesilencing reagents) (refer to “Custom gripNA™ Synthesis Service”handbook (version B2, available through ActiveMotif atwww.activemotif.com) and to U.S. Pat. No. 6,962,906, incorporated in itsentirety herein by reference).

In additional embodiments, the oligonucleotide reagents of the inventioncan be generated as steroid-conjugated PNAs. For example, a steroid(e.g., glucocorticoid) dexamethasone can be linked to a PNA of theinstant invention, as described in Rebuffat, A. G., et al. (FASEB J.2002 16(11):1426-8, the entire contents of which are incorporated hereinby reference). The oligonucleotide reagents of the invention can also beproduced as tricycle-DNA molecules ((tc)-DNAs) that are ISS-N1 splicesite-targeted, as described in Ittig, D., et al. (Nucleic Acids Res.2004 32(1):346-53, the entire contents of which are incorporated hereinby reference).

The oligonucleotide reagents of the invention can also be formulated asmorpholino oligonucleotides. In such embodiments, the riboside moiety ofeach subunit of an oligonucleotide of the oligonucleotide reagent isconverted to a morpholine moiety (morpholine=C₄H₉NO; refer to Heasman,J. 2002 Developmental Biology 243, 209-214, the entire contents of whichare incorporated herein by reference).

The preceding forms of modifications can improve the delivery and/oractivity of the oligonucleotide reagents of the invention, or also canbe used to minimize non-specific effects potentially associated withalternative formulations of the oligonucleotide reagents of the instantinvention.

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acid moleculeshaving at least one region which is complementary to a nucleic acidmolecule of the invention, such that the molecular beacon is useful forquantitating the presence of the nucleic acid molecule of the inventionin a sample. A “molecular beacon” nucleic acid is a nucleic acidmolecule comprising a pair of complementary regions and having afluorophore and a fluorescent quencher associated therewith. Thefluorophore and quencher are associated with different portions of thenucleic acid in such an orientation that when the complementary regionsare annealed with one another, fluorescence of the fluorophore isquenched by the quencher. When the complementary regions of the nucleicacid molecules are not annealed with one another, fluorescence of thefluorophore is quenched to a lesser degree. Molecular beacon nucleicacid molecules are described, for example, in U.S. Pat. No. 5,876,930.

In another embodiment, oligonucleotide reagents of the invention containsequences which naturally flank the ISS-N1 sequence (i.e., sequenceslocated at the 5′ and 3′ ends of the ISS-N1 sequence) in the genomic DNAof an organism. In various embodiments, the isolated oligonucleotideagent can contain about 100 kB, 50 kB, 25 kB, 15 kB, 10 kB, 5 kB, 4 kB,3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the ISS-N1 sequence in genomic DNA of the targeted cell.Moreover, an oligonucleotide reagent can be substantially free of othercellular material or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

The target RNA (e.g., pre-mRNA) blocking reaction guided byoligonucleotide reagents of the invention is highly sequence specific.In general, oligonucleotide reagents containing nucleotide sequencesperfectly complementary to a portion of the target RNA are preferred forblocking of the target RNA. However, 100% sequence complementaritybetween the oligonucleotide reagent and the target RNA is not requiredto practice the present invention. Thus, the invention may toleratesequence variations that might be expected due to genetic mutation,strain polymorphism, or evolutionary divergence. For example,oligonucleotide reagent sequences with insertions, deletions, and singlepoint mutations relative to the target sequence may also be effectivefor inhibition. Alternatively, oligonucleotide reagent sequences withnucleotide analog substitutions or insertions can be effective forblocking.

Greater than 70% sequence identity (or complementarity), e.g., 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the oligonucleotide reagent and thetarget RNA, e.g., target pre-mRNA, is preferred.

In addition, variants of the ISS-N1 sequence which retain the functionof ISS-N1 can be used in the methods of the invention. For example, aseries of mutants of ISS-N1 were generated in Example 14 and tested fortheir ability to inhibit alternative splicing. Each of the mutant formswas found to retain ISS-N1 activity. In one embodiment, such variantsequences are at least about 95% identical in sequence to ISS-N1 overthe entire length of the ISS-N1 15 nucleotide sequence. In anotherembodiment, such variant sequences are at least about 90% identical insequence to ISS-N1 over the entire length of the ISS-N1 15 nucleotidesequence. In another embodiment, such variant sequences are at leastabout 85% identical in sequence to ISS-N1 over the entire length of theISS-N1 15 nucleotide sequence. In another embodiment, such variantsequences are at least about 80% identical in sequence to ISS-N1 overthe entire length of the ISS-N1 15 nucleotide sequence. In anotherembodiment, such variant sequences are at least about 75% identical insequence to ISS-N1 over the entire length of the ISS-N1 15 nucleotidesequence. In another embodiment, such variant sequences are at leastabout 70% identical in sequence to ISS-N1 over the entire length of theISS-N1 15 nucleotide sequence. In another embodiment, such variantsequences are at least about 66% identical in sequence to ISS-N1 overthe entire length of the ISS-N1 15 nucleotide sequence.

Sequence identity, including determination of sequence complementarityfor nucleic acid sequences, may be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Alternatively, the oligonucleotide reagent may be defined functionallyas a nucleotide sequence (or oligonucleotide sequence) a portion ofwhich is capable of hybridizing with the target RNA (e.g., 400 mM NaCl,40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16hours; followed by washing). Additional preferred hybridizationconditions include hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC,50% formamide followed by washing at 70° C. in 0.3×SSC or hybridizationat 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washingat 67° C. in 1×SSC. The hybridization temperature for hybridsanticipated to be less than 50 base pairs in length should be 5-10° C.less than the melting temperature (Tm) of the hybrid, where Tm isdetermined according to the following equations. For hybrids less than18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases).For hybrids between 18 and 49 base pairs in length, Tm(°C.)=81.5+16.6(log 10[Na+])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([Na+] for 1×SSC=0.165 M). Additional examplesof stringency conditions for polynucleotide hybridization are providedin Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11, and Current Protocols inMolecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons,Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. Thelength of the identical nucleotide sequences may be at least about 10,12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

Modifications

In a preferred aspect, the oligonucleotide reagents (e.g,oligoribonucleotides, such as anti-ISS-N1 oligoribonucleotides) of thepresent invention are modified to improve stability in serum or growthmedium for cell cultures, or otherwise to enhance stability duringdelivery to SMA subjects and/or cell cultures. In order to enhance thestability, the 3′-residues may be stabilized against degradation, e.g.,they may be selected such that they consist of purine nucleotides,particularly adenosine or guanosine nucleotides. Alternatively,substitution of pyrimidine nucleotides by modified analogues, e.g.,substitution of uridine by 2′-deoxythymidine can be tolerated withoutaffecting the efficiency of oligonucleotide reagent-induced modulationof splice site selection. For example, the absence of a 2′ hydroxyl maysignificantly enhance the nuclease resistance of the oligonucleotidereagents in tissue culture medium.

In an especially preferred embodiment of the present invention theoligonucleotide reagents, e.g., anti-ISS-N1 antisense molecules, maycontain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificactivity, e.g., the splice site selection modulating activity is notsubstantially effected, e.g., in a region at the 5′-end and/or the3′-end of the oligonucleotide (in preferred embodiments,oligoribonucleotide) molecule. Particularly, the ends may be stabilizedby incorporating modified nucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.Oligonucleotide reagents of the invention also may be modified withchemical moieties (e.g., cholesterol) that improve the in vivopharmacological properties of the oligonucleotide reagents.

A further preferred oligonucleotide modification includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226, theentire contents of which are incorporated by reference herein.

Within the oligonucleotide reagents (e.g., oligoribonucleotides) of theinvention, as few as one and as many as all nucleotides of theoligonucleotide can be modified. For example, a 20-mer oligonucleotidereagent (e.g., oligoribonucleotide) of the invention may contain 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20modified nucleotides. In preferred embodiments, the modifiedoligonucleotides (e.g., oligoribonucleotides) of the invention willcontain as few modified nucleotides as are necessary to achieve adesired level of in vivo stability and/or bioaccessibility whilemaintaining cost effectiveness.

RNA molecules and oligonucleotide reagents may be produced enzymaticallyor by partial/total organic synthesis, any modified ribonucleotide canbe introduced by in vitro enzymatic or organic synthesis. In oneembodiment, an RNA molecule, e.g., oligonucleotide reagent, is preparedchemically. Methods of synthesizing RNA and DNA molecules are known inthe art, in particular, the chemical synthesis methods as described inVerma and Eckstein (1998) Annul Rev. Biochem. 67:99-134. RNA can bepurified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. Alternatively,the RNA molecules, e.g., oligonucleotide reagents, can also be preparedby enzymatic transcription from synthetic DNA templates or from DNAplasmids isolated from recombinant bacteria. Typically, phage RNApolymerases are used such as T7, T3 or SP6 RNA polymerase (Milligan andUhlenbeck (1989) Methods Enzymol. 180:51-62). The RNA may be dried forstorage or dissolved in an aqueous solution. The solution may containbuffers or salts to inhibit annealing, and/or promote stabilization ofthe single strands.

In preferred embodiments of the invention, the target RNA of anoligonucleotide reagent specifies the amino acid sequence of SMNprotein. As used herein, the phrase “specifies the amino acid sequence”of a SMN means that the mRNA sequence is translated into a SMN aminoacid sequence according to the rules of the genetic code.

By blocking domains within RNAs (e.g., pre-mRNAs) capable of beingtranslated into such proteins, valuable information regarding thefunction of said oligonucleotide reagent and/or proteins and therapeuticbenefits of said blocking may be obtained.

Splice forms and expression levels of surveyed RNAs and proteins may beassessed by any of a wide variety of well known methods for detectingsplice forms and/or expression of a transcribed nucleic acid or protein.Non-limiting examples of such methods include RT-PCR of spliced forms ofRNA followed by size separation of PCR products, nucleic acidhybridization methods e.g., Northern blots and/or use of nucleic acidarrays; nucleic acid amplification methods; immunological methods fordetection of proteins; protein purification methods; and proteinfunction or activity assays.

RNA expression levels can be assessed by preparing mRNA/cDNA (i.e. atranscribed polynucleotide) from a cell, tissue or organism, and byhybridizing the mRNA/cDNA with a reference polynucleotide which is acomplement of the assayed nucleic acid, or a fragment thereof. cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction or in vitro transcription methods prior to hybridization withthe complementary polynucleotide; preferably, it is not amplified.Expression of one or more transcripts can also be detected usingquantitative PCR to assess the level of expression of the transcript(s).

In one embodiment, oligonucleotide reagents are synthesized either invivo, in situ, or in vitro. Endogenous RNA polymerase of the cell maymediate transcription in vivo or in situ, or cloned RNA polymerase canbe used for transcription in vivo or in vitro. For transcription from atransgene in vivo or an expression construct, a regulatory region (e.g.,promoter, enhancer, silencer, splice donor and acceptor,polyadenylation) may be used to transcribe the oligonucleotide reagent.Production of oligonucleotide reagents may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. A transgenic organism that expresses an oligonucleotide reagentfrom a recombinant construct may be produced by introducing theconstruct into a zygote, an embryonic stem cell, or another multipotentcell derived from the appropriate organism.

II. DNA Cassettes

In certain aspects of the invention, DNA cassettes can be generated todisplace and/or disrupt the ISS-N1 region via homologous recombination.Displacement and/or disruption of the ISS-N1 region in a cell can beperformed by art-recognized techniques, such as chemical delivery,electroporation and/or retroviral delivery of DNA cassettes. Such DNAcassettes may be delivered to a cell in vivo or ex vivo, e.g., toneuronal and/or embryonic stem cells, which may then be reintroducedinto a subject for therapeutic purpose.

III. Methods of Introducing RNAs, Vectors, and Host Cells

An oligonucleotide reagent construct of the present invention can bedelivered to cells ex vivo or in vivo, for example, as an expressionplasmid which, when transcribed in the cell, produces RNA, which iscomplementary to at least a unique portion of the cellular pre-mRNAwhich encodes an SMN protein.

Alternatively, the oligonucleotide reagent can be an oligonucleotidewhich is generated ex vivo and which, when introduced into the cell,causes inhibition of expression by hybridizing with the pre-mRNA, mRNAand/or genomic sequences of the SMN2 gene. Such oligonucleotides arepreferably modified oligonucleotides, which are resistant to endogenousnucleases, e.g. exonucleases and/or endonucleases, and are thereforestable in vivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of oligonucleotide (see also U.S. Pat. Nos.5,176,996, 5,294,564 and 5,256,775, which are herein incorporated byreference).

Oligonucleotide sequences can be introduced into cells as is known inthe art. Transfection, electroporation, fusion, liposomes, colloidalpolymeric particles and viral and non-viral vectors as well as othermeans known in the art may be used to deliver the oligonucleotidesequences to the cell. The method of delivery selected will depend atleast on the cells to be treated and the location of the cells and willbe known to those skilled in the art. Localization can be achieved byliposomes, having specific markers on the surface for directing theliposome, by having injection directly into the tissue containing thetarget cells, by having depot associated in spatial proximity with thetarget cells, specific receptor mediated uptake, viral vectors, or thelike.

In certain embodiments, ribozymes can be used to deliver oligonucleotidereagents of the invention directed against ISS-N1 sequences (includingfunctional variants of ISS-N1 sequence) to a necessary site within agiven intron. Ribozyme design is an art-recognized process, described,e.g., in U.S. Pat. No. 6,770,633, the entire contents of which areincorporated by reference herein.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

As described supra and in the art, oligonucleotide reagents may bedelivered using, e.g., methods involving liposome-mediated uptake, lipidconjugates, polylysine-mediated uptake, nanoparticle-mediated uptake,and receptor-mediated endocytosis, as well as additional non-endocyticmodes of delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (refer to Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44, 35-49, incorporated in its entiretyherein by reference).

Oligonucleotide reagents may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the RNA using methods known in the art for introducingnucleic acid (e.g., DNA) into cells in vivo. Vascular or extravascularcirculation, the blood or lymph system, and the cerebrospinal fluid aresites where the RNA may be introduced.

The present invention also provides vectors comprising an expressioncontrol sequence operatively linked to the oligonucleotide sequences ofthe invention. The present invention further provides host cells,selected from suitable eucaryotic and procaryotic cells, which aretransformed with these vectors as necessary. Such transformed cellsallow the study of the function and the regulation of malignancy and thetreatment therapy of the present invention.

Vectors are known or can be constructed by those skilled in the art andshould contain all expression elements necessary to achieve the desiredtranscription of the sequences. Other beneficial characteristics canalso be contained within the vectors such as mechanisms for recovery ofthe oligonucleotides in a different form. Phagemids are a specificexample of such beneficial vectors because they can be used either asplasmids or as bacteriophage vectors. Examples of other vectors includeviruses such as bacteriophages, baculoviruses and retroviruses, DNAviruses, liposomes and other recombination vectors. The vectors can alsocontain elements for use in either procaryotic or eucaryotic hostsystems. One of ordinary skill in the art will know which host systemsare compatible with a particular vector. The vectors can be introducedinto cells or tissues by any one of a variety of known methods withinthe art. Such methods can be found generally described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (1989, 1992), in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Changet al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vegaet al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988) and Gilboa et al., BioTechniques 4:504-512 (1986) andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Viralvectors that have been used for gene therapy protocols include, but arenot limited to, retroviruses, other RNA viruses such as poliovirus orSindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV40, vaccinia and other DNA viruses. Replication-defective murineretroviral vectors are the most widely utilized gene transfer vectors.Murine leukemia retroviruses are composed of a single strand RNAcompleted with a nuclear core protein and polymerase (pol) enzymesencased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include gag, pol, and env genes enclosed at the 5′ and 3′long terminal repeats (LTRs). Retroviral vector systems exploit the factthat a minimal vector containing the 5′ and 3′ LTRs and the packagingsignal are sufficient to allow vector packaging and infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line.

Recombinant methods known in the art can also be used to achieveoligonucleotide reagent-induced inhibition of splicing in a targetnucleic acid. For example, vectors containing oligonucleotide reagentscan be employed to express, e.g., an antisense oligonucleotide toinhibit splicing of an exon of a targeted pre-mRNA.

Examples of methods to introduced oligonucleotide sequences into cellsencompass both non-viral and viral methods, as well as in vivo and exvivo methods and include, for example:

Direct Injection: Naked DNA can be introduced into cells in vivo bydirectly injecting the DNA into the cells (see e.g., Acsadi et al.(1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).For example, a delivery apparatus (e.g., a “gene gun”) for injecting DNAinto cells in vivo can be used. Such an apparatus is commerciallyavailable (e.g., from BioRad).

Cationic Lipids Naked DNA can be introduced into cells in vivo bycomplexing the DNA with cationic lipids or encapsulating the DNA incationic liposomes. Examples of suitable cationic lipid formulationsinclude N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride(DOTMA) and a 1:1 molar ratio of1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE)and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. etal. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human GeneTherapy 4:781-788).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids which naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126). Carrier mediated gene transfer may alsoinvolve the use of lipid-based compounds which are not liposomes. Forexample, lipofectins and cytofectins are lipid-based positive ions thatbind to negatively charged DNA and form a complex that can ferry the DNAacross a cell membrane. Another method of carrier mediated gene transferinvolves receptor-based endocytosis. In this method, a ligand (specificto a cell surface receptor) is made to form a complex with a gene ofinterest and then injected into the bloodstream. Target cells that havethe cell surface receptor will specifically bind the ligand andtransport the ligand-DNA complex into the cell.

Retroviruses: Defective retroviruses are well characterized for use ingene transfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). A recombinant retrovirus can be constructed havinga nucleotide sequences of interest incorporated into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ψ Crip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Retroviral vectors requiretarget cell division in order for the retroviral genome (and foreignnucleic acid inserted into it) to be integrated into the host genome tostably introduce nucleic acid into the cell. Thus, it may be necessaryto stimulate replication of the target cell.

Adenoviruses: The genome of an adenovirus can be manipulated such thatit encodes and expresses a gene product of interest but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use are deletedfor all or parts of the viral E1 and E3 genes but retain as much as 80%of the adenoviral genetic material.

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product.

In a preferred embodiment, a retroviral expression vector encoding anoligonucleotide of the invention is used in vivo, to thereby inhibit theactivity of the ISS-N1 domain of SMN2, and thus promote SMN2 exon 7inclusion in vivo. Such retroviral vectors can be prepared according tostandard methods known in the art.

A modulatory agent, such as a chemical compound, can be administered toa subject as a pharmaceutical composition. Such compositions typicallycomprise the modulatory agent and a pharmaceutically acceptable carrier.As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. Pharmaceutical compositions can beprepared as described below in subsection IV.

Cells targeted or used in the methods of the instant invention arepreferably mammalian cells, in particular, human cells. Cells may befrom the germ line or somatic, totipotent or pluripotent, dividing ornon-dividing, parenchyma or epithelium, immortalized or transformed, orthe like. The cell may be a stem cell or a differentiated cell. Celltypes that are differentiated include adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine orexocrine glands. Neurons and muscle cells (e.g., myocytes, myoblasts,myotubes, myofibers, and the like) are preferred target cells of theinvention.

Depending on the particular target gene and the dose of oligonucleotidereagent material delivered, this process may modulate function of thetarget gene. In exemplary embodiments of the instant invention, exon7-containing SMN protein production is enhanced in a treated cell, cellextract, organism or patient, with an enhancement of exon 7-containingSMN protein levels of at least about 1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-,7-, 10-, 20-, 100-fold and higher values being exemplary. Enhancement ofgene expression refers to the presence (or observable increase) in thelevel of protein and/or mRNA product from a target RNA. Specificityrefers to the ability to act on the target RNA without manifest effectson other genes of the cell. The consequences of modulation of the targetRNA can be confirmed by examination of the outward properties of thecell or organism (as presented below in the examples) or by biochemicaltechniques such as RNA solution hybridization, nuclease protection,Northern hybridization, reverse transcription, gene expressionmonitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS).

For oligonucleotide reagent-mediated modulation of an RNA in a cell lineor whole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereof.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin. Depending on the assay, quantitation of the amount of geneexpression allows one to determine a degree of modulation which isgreater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell nottreated according to the present invention. Lower doses of injectedmaterial and longer times after administration of oligonucleotidereagents may result in modulation in a smaller fraction of cells (e.g.,at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells).Quantitation of gene expression in a cell may show similar amounts ofmodulation at the level of accumulation of target mRNA or translation oftarget protein. As an example, the efficiency of modulation may bedetermined by assessing the amount of gene product in the cell; pre-mRNAor mRNA may be detected with a hybridization probe having a nucleotidesequence outside the region used for the oligonucleotide reagent, ortranslated polypeptide may be detected with an antibody raised againstthe polypeptide sequence of that region.

The oligonucleotide reagent may be introduced in an amount which allowsdelivery of at least one copy per cell. Higher doses (e.g., at least 5,10, 100, 500 or 1000 copies per cell) of material may yield moreeffective modulation; lower doses may also be useful for specificapplications.

IV. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted target geneexpression or activity (e.g., in exemplary embodiments, underexpressionof SMN protein). “Treatment”, or “treating” as used herein, is definedas the application or administration of a therapeutic agent (e.g., anoligonucleotide reagent (e.g., oligoribonucleotide) or vector ortransgene encoding same, a small molecule ISS-N1 blocking agent, etc.)to a patient, or application or administration of a therapeutic agent toan isolated tissue or cells (including fetal cells) from a patient, whohas a disease or disorder, a symptom of disease or disorder or apredisposition toward a disease or disorder, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease or disorder, the symptoms of the disease or disorder, or thepredisposition toward disease.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject atherapeutic agent (e.g., an oligonucleotide reagent (e.g.,oligoribonucleotide) or vector or transgene encoding same, a smallmolecule ISS-N1 blocking agent, etc.). Subjects at risk for a diseasewhich is caused or contributed to by aberrant or unwanted target geneexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the target gene aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of target gene aberrancy, forexample, a target gene, target gene agonist or target gene antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating targetgene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent (e.g., an oligonucleotide reagent (e.g.,oligoribonucleotide) or vector or transgene encoding same, a smallmolecule ISS-N1 blocking agent, etc.) that is specific for the targetgene or protein (e.g., is specific for the pre-mRNA encoded by said geneand/or specifying the amino acid sequence of said protein) such thatexpression or one or more of the activities of target protein ismodulated. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant or unwanted expression or activity ofa target gene polypeptide or nucleic acid molecule. Modulation of targetgene activity is desirable in situations in which target gene isabnormally unregulated and/or in which altered target gene activity islikely to have a beneficial effect.

In one embodiment, cells from a subject having spinal muscular atrophyare contacted with an oligonucleotide reagent of the invention toinhibit splicing of the SMN2 exon 7. Exemplary oligonucleotide reagentsinclude sequences complementary to the ISS-N1 sequence and variantsthereof (e.g., as shown in Example 14). In another embodiment, cellsfrom a subject having another disorder that would benefit frominhibition of alternative splicing are contacted with an oligonucleotidereagent of the invention. Target sequences related to the ISS-N1sequence are present in human intronic sequences. For example, there isa sequence partially homologous to the ISS-N1 -sequence located in humanCFTR (intron 10). Additional exemplary genes that can be targeted byoligonucleotide reagents of the invention (e.g., sequences complementaryto the ISS-N1 sequence and variants thereof (e.g., as shown in Example14) include, but are not limited to, CFTR, FAS, Caspases, Diablo, NF1,Bc12, Tau, ApoA-11, p53, Tra2, Cox-1 and Survivin.

3. Delivery of Oligonucleotide Reagents to the Nervous System

The oligonucleotide reagents of the invention can be delivered to thenervous system of a subject by any art-recognized method. For example,peripheral blood injection of the oligonucleotide reagents of theinvention can be used to deliver said reagents to peripheral neurons viadiffusive and/or active means. Alternatively, the oligonucleotidereagents of the invention can be modified to promote crossing of theblood-brain-barrier (BBB) to achieve delivery of said reagents toneuronal cells of the central nervous system (CNS). Specific recentadvancements in oligonucleotide reagent technology and deliverystrategies have broadened the scope of oligonucleotide reagent usage forneuronal disorders (Forte, A., et al. 2005. Curr. Drug Targets 6:21-29;Jaeger, L. B., and W. A. Banks. 2005. Methods Mol. Med. 106:237-251;Vinogradov, S. V., et al. 2004. Bioconjug. Chem. 5:50-60; the precedingare incorporated herein in their entirety by reference). For example,the oligonucleotide reagents of the invention can be synthesized tocomprise phosphorothioate oligodeoxynucleotides (P-ODN) directed againstISS-N1, or may be generated as peptide nucleic acit (PNA) compounds.P-ODN and PNA reagents have each been identified to cross the BBB(Jaeger, L. B., and W. A. Banks. 2005. Methods Mol. Med. 106:237-251).Treatment of a subject with, e.g., a vasoactive agent, has also beendescribed to promote transport across the BBB (ibid.). Tethering of theoligonucleotide reagents of the invention to agents that are activelytransported across the BBB may also be used as a delivery mechanism(ibid.).

In certain embodiments, the oligonucleotide reagents of the inventioncan be delivered by transdermal methods (e.g., via incorporation of theoligonucleotide reagent(s) of the invention into, e.g., emulsions, withsuch oligonucleotide reagents optionally packaged into liposomes). Suchtransdermal and emulsion/liposome-mediated methods of delivery aredescribed for delivery of antisense oligonucleotides in the art, e.g.,in U.S. Pat. No. 6,965,025, the contents of which are incorporated intheir entirety by reference herein.

The oligonucleotide reagents of the invention may also be delivered viaan implantable device (e.g., pacemaker or other such implantabledevice). Design of such a device is an art-recognized process, with,e.g., synthetic implant design described in, e.g., U.S. Pat. No.6,969,400, the contents of which are incorporated in their entirety byreference herein.

4. Pharmacogenomics

The therapeutic agents (e.g., an oligonucleotide reagent or vector ortransgene encoding same) of the invention can be administered toindividuals to treat (prophylactically or therapeutically) disordersassociated with aberrant or unwanted target gene activity. Inconjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) may be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a targetgene polypeptide of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a therapeutic agent of thepresent invention can give an indication whether gene pathways relatedto toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a therapeuticagent, as described herein.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an oligonucleotide reagent (or expression vector or transgeneencoding same) as described herein can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with saidagent. Alternatively, a therapeutic agent can be used in an animal modelto determine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

V. Pharmaceutical Compositions

The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsof the present invention (e.g., oligonucleotides, small molecules andthe like) can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, antibody, or modulatory compound and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds that exhibit largetherapeutic indices are preferred. Although compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects. The data obtained from the cell culture assays andanimal studies can be used in formulating a range of dosage for use inhumans. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES

The following materials, methods, and examples are illustrative only andnot intended to be limiting.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, and standard techniques inelectrophoresis. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989) and CurrentProtocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons(1992).

Minigenes. Minigene splicing cassettes pSMN1ΔI6 and pSMN2ΔI6 wereconstructed by deleting approximately 6 kb within intron 6 from pSMN1and pSMN2 , respectively (Singh, N.N., et al. 2004. Biochem. Biophys.Res. Commun. 315:381-388). In certain of the following Examples, SMN1and SMN2 refer to pSMN1ΔI6 and pSMN2 ΔI6, respectively, describedpreviously (ibid.). Mutations were generated by PCR using the strategydescribed in Singh, N.N., et al. 2004. Biochem. Biophys. Res. Commun.315:381-388. Minigene splicing cassette Casp3 was constructed byamplifying genomic sequences spanning exon 5 to exon 7 of the Caspase 3gene, using high fidelity Pfx polymerase (Invtrogen), genomic DNA(Clontech) and a pair of primers P53 (GTCCTCGAGTTTCTAAAGAAGATCACAGC, SEQID NO: 7) and P56 (GTCGCGGCCGCACCATCTTCTCACTTGGCAT, SEQ ID NO: 8). Theresulting PCR fragment was subsequently digested with Xho I and Not I(NEB) and cloned into pCI vector (Promega). Minigene splicing cassetteCasp3Avr was generated by inserting an Avr II restriction site (CCTAGG)in Casp3 minigene downstream of an alternatively-spliced exon 6 usinghigh fidelity PCR. Minigene splicing cassette Casp3ISS-N1 was generatedby inserting an ISS-N1 sequence (CCAGCATTATGAAAG, SEQ ID NO: 9) usingAvr II restriction site in Casp3Avr. The exact locations of Avr IIand/or ISS-N1 sites are shown in FIG. 11C. Splicing cassettespTBEx9-V456F (CFTR exon 9 splicing), pTBEx12-50A (CFTR exon 12 splicing)and pTBApo-ISE3m (apoA-II exon 3 of splicing) were the same as inMercado, P.A., et al. 2005. Nude. Acids Res. (in Press); Pagani, F., etal. 2003. J. Biol. Chem. 278:26580-26588; Pagani, F., et al. 2003. Hum.Mol. Genet. 12:1111-1120. Splicing cassettes CMV-Fas (wt) and CMV-Fasmutant (U-20C) (Fas exon 6 splicing) were the same as in Izquierdo,J.M., et al. 2005. Mol. Cell 19:475-484. Splicing cassette SI9/LI10 (Tauexon 10 splicing) was the same as in Yu, Q., et al. 2004. J. Neurochem.90:164-72.

Cell culture. Unless otherwise stated, all tissue culture media andsupplements were purchased from Invitrogen. Human cervical carcinoma(C33a) cells, human HEK-293 cells and mouse motor-neuron-like (NSC-34)cells were cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillinand 100 μg/ml streptomycin. Human neuroblastoma (SK-N-SH) cells weremaintained in 1:1 mixture of Minimum Essential Medium (MEM) and Ham'sF12 medium supplemented with 10% FBS. Mouse neuroblastoma (Neuro-2a)cells were grown in MEM supplemented with 2 mM L-glutamine, 0.1 mMnon-essential amino acids, 1.0 mM sodium pyruvate and 10% FBS. Mouseteratocarcinoma (P19) cells were maintained in alpha-MEM supplementedwith 10% FBS. All cells mentioned above were obtained from the AmericanType Culture Collection. NSC-34 cells were obtained from Dr. NeilCashman (University of Toronto). Primary fibroblast cell line from SMAtype I patient (GM03813) and a healthy control (AG06814) were obtainedfrom Coriell Cell Repositories. These cell lines were maintained in MEMsupplemented with 2 mM L-glutamine and 15% FBS.

Anti-N1: (SEQ ID NO: 10)5′-mA*mU*mU*mC*mA*mC*mU*mU*mU*mC*mA*mU*mA*mA*mU* mG*mC*mU*mG*mG3′;Anti-N1 + 10: (SEQ ID NO: 11)5′-mC*mA*mA*mA*mA*mG*mU*mA*mA*mG*mA*mU*mU*mC*mA* mC*mU*mU*mU*mC3′;Anti-N1 + 20: (SEQ ID NO: 12)5′-mU*mA*mA*mA*mG*mU*mU*mU*mU*mA*mC*mA*mA*mA*mA* mG*mU*mA*mA*mG3′;Anti-N1 + 30: (SEQ ID NO: 13)5′-mC*mC*mA*mC*mA*mA*mA*mC*mC*mA*mU*mA*mA*mA*mG* mU*mU*mU*mU*mA3′;Scramble20: (SEQ ID NO: 14)5′-mU*mC*mC*mU*mU*mU*mA*mA*mA*mG*mU*mA*mU*mU*mG* mU*mG*mA*mC*mC3′;Anti-17-25: (SEQ ID NO: 15)5′-mA*mU*mU*mC*mA*mC*mU*mU*mU*mC*mU*mA*mA*mA*mU* mU*mA*mA*mG*mG3′;Anti-ISS-N1/15: (SEQ ID NO: 16)5′-mC*mU*mU*mU*mC*mA*mU*mA*mA*mU*mG*mC*mU*mG*mG-3′

The abbreviation “m” represents O-methyl modification at the 2ndposition of sugar residue, whereas “*” represents a phosphorothioatemodification of the backbone. Scramble20 and Anti-N1 had the samesequence composition: three guanosines, four cytosines, five adenosinesand eight uridines.

Transfections and in vivo splicing assays. All reagents were usedaccording to manufacturer's recommendations. Transient transfections ofcells with plasmid DNA or antisense oligonucleotides were performedusing Lipofectamine™ 2000 (Invitrogen). Cells were plated 24 hours priorto transfection so that their density on the day of transfection wasapproximately 90%. For co-transfection experiments, cells weretransfected with the indicated amounts of plasmid DNA and anoligonucleotide of interest. Oligonucleotide concentration ranged from25 to 500 nM. In a given experiment, the total amount of oligonucleotidewas maintained constant by adding control oligonucleotide (Scramble20).Unless indicated otherwise, total RNA was isolated 24 hours aftertransfection using Trizol™ reagent (Invitrogen). To generate cDNA,reverse-transcription was carried out using the SuperScript™ II reactionkit (Invitrogen). Oligo (dT) primer was used in case of pCI-basedminigenes, while random hexamers and vector-specific 3′ primer PT2(5′AAGCTTGCATCGAATCAGTAG3′, SEQ ID NO: 17) were used in the case ofpTB-vector-based minigenes and Fas minigenes, respectively. Generally,1.0 μg of total RNA was used per 20 μl of reaction. Minigene-specificspliced products were subsequently identified using Taq polymerase(Invitrogen) and the following primer combinations: P1(5′CGACTCACTATAGGCTAGCC3′, SEQ ID NO: 18) and P25′GCATGCAAGCTTCCTTTTTTCTTTCCCAACAC3′, SEQ ID NO: 19) for SMN minigenes(Singh, N.N., et al. 2004. Biochem. Biophys. Res. Commun. 315:381-388);alfa-23 (5′CAACTTCAAGCTCCTAAGCCACTGC3′, SEQ ID NO: 20) and BRA2(5′TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA3′ (SEQ ID NO: 21) for CFTR andapoA-II minigenes (Mercado, P.A., et al. 2005. Nucle. Acids Res. (inPress); Pagani, F., et al. 2003. J. Biol. Chem. 278:26580-26588; Pagani,F., et al. 2003. Hum. Mol. Genet. 12:1111-1120); FP(5′GGTGTCCACTCCCAGTTCAA3′, SEQ ID NO: 22) and RP(5′CCCTGGTTTATGATGGATGTTGCCTAATGAG3′, SEQ ID NO: 23) for Tau minigene(Yu, Q., et al. 2004. J. Neurochem. 90:164-72); P1 and P56 for Casp3minigenes; PT1 (5′GTCGACGACACTTGCTCAAC3′, SEQ ID NO: 24) and PT2 for Fasminigenes (Izquierdo, J.M., et al. 2005. Mol. Cell 19:475-484). Analysisand quantifications of spliced products were performed using a FPL-5000Image Reader and ImageGauge software (Fuji Photo Film Inc.; Singh, N.N.,et al. 2004. Biochem. Biophys. Res. Commun. 315:381-388). Results wereconfirmed by three independent experiments.

Expression of endogenous genes. For GM03813 and AG06814 fibroblasttransfection, cells were plated at a density of ˜0.3×10⁵ per one well ofa 24-well plate one day before transfection. Cells were then transfectedwith an indicated oligonucleotide (from 5 to 200 nM). Totaloligonucleotide amount was held constant by adding scrambledoligonucleotide (Scramble20). Unless indicated otherwise, total RNA wasisolated 24 hours after transfection using Trizol reagent (Invitrogen).Reverse-transcription was carried out using oligo (dT) primer andSuperScript™ II (Invitrogen) as described above. For PCR amplificationof endogenous exons, the following primer combinations were used: N-24(5′CCAGATTCTCTTGATGATGCTGATGCTTTGGG3′, SEQ ID NO: 25) and P2(5′GCATGCAAGCTTCCTTTTTTCTTTCCCAACAC3′, SEQ ID NO: 26) for SMN exon 7;Ex4Sense (5′CGGAATTCCAATGAAAATGAAAGCCAAGTTTCAAC3′, SEQ ID NO: 27) andEx6Anti (5′ATAGTTTAGCGGCCGCCATATAATAGCCAGTATGATAG3′, SEQ ID NO: 28) forSMN exon 5; Ex1Sense (5′CGGAATTCCATGGCGATGAGCAGCGGCGGCAG3′, SEQ ID NO:29) and Ex4Anti (5′ATAGTTTAGCGGCCGCCTTTCCTGGTCCCAGTCTTGG3′, SEQ ID NO:30) for SMN exon 3; P53 and P56 for Caspase 3 exon 6; 5′-Sur(GCATGGGTGCCCCGACGTTG, SEQ ID NO: 31) and 3′-Sur (GCTCCGGCCAGAGGCCTCAA,SEQ ID NO: 32) for Survivin exons 2B/3 (Mahotka, C., et al. 1999. CancerRes. 59:6097-6102); A1 (CATGAGCGACAGCGGCGAGCAGAA, SEQ ID NO: 33) and A3(TTAATAGCGACGAGGTGAGTA, SEQ ID NO: 34) for Tra2 exons 2 a/2 b (Chen, X.,et al. 2003. Cell Biol. Int. 27:491-496); 5′Bcl-X (CATGGCAGCAGTAAAGCAAG,SEQ ID NO: 35) and 3′Bcl-X (GCATTGTTCCCATAGAGTTCC, SEQ ID NO: 36) forBcl-x exon 2 (Mercatante, D.R., et al. 2002. J. Biol Chem.277:49374-49382); ex28F (GGAGTACACCAAGTATCATGAG, SEQ ID NO: 37) andex31R (CATTATGCTTGCAAAAACGAAC, SEQ ID NO: 38) for NF1 exons 29/30 (Park,V.M., et al. 1998. Hum. Genet. 103:382-385). Results were confirmed bythree independent experiments.

Antibodies and western blot analysis. Transfections with antisenseoligonucleotides were done as described above. For each sample, the samenumber of cells (˜9×10⁵) was harvested 72 hours after transfection andlysates were prepared as in Elbashir, S. M., et al. 2002. Methods26:199-213. One third of each lysate was used for one blot. Lysates wereelectrophoresed on a 10% (w/v) SDS-PAGE gel and the proteins weretransferred onto polyvinylidene fluoride (PVDF) membrane (Pall LifeSciences). The protein transfer and equal loading were verified by SYPRORuby protein blot staining (Bio-Rad Laboratories). The membranes wereblocked with 5% non-fat dried milk in TBST (20 mM Tris, 150 mM NaCl and0.1% Tween 20) overnight at 4° C. and subsequently incubated withprimary anti-SMN antibodies (BD Transduction Laboratories) followed bywashing and incubation with horseradish peroxidase-conjugated goatanti-mouse IgG (Jackson Immunoresearch). For a loading control,membranes were stripped with buffer containing 10 mM Tris, 75 mM NaCl,1% SDS and 10 mM β-mercaptoethanol (30 minutes at 60° C.) and re-probedfor α-tubulin. Monoclonal antibodies against α-tubulin were from Sigma.Immunoreactive proteins were visualized using SuperSignal West DuraExtended Duration Substrate (Pierce). The membranes were scanned using aLAS-1000 Image Reader (Fuji Photo Film Inc.). Results were confirmed bythree independent experiments.

Example 1 In Vivo Selection of the Entire Exon 7 Revealed NovelCis-Elements

Understanding the mechanism of splicing of exon 7 in SMN genes isimportant for development of mechanism-based therapeutic strategies.Towards this goal, the entire exon 7 of SMN2 was examined using a stateof the art method of in vivo selection (the selection process wasperformed on a pool of minigene sequences comprising partiallyrandomized exon 7 sequences, with iterative selection across individualrounds of PCR and size separation for exon 7-containing SMN2 mutants;for further details, refer to Singh et al., RNA 10, 1291-1305). Threemajor cis-acting elements were identified by this selection process(termed Exinct, Conserved tract, and 3′—Cluster; refer to FIG. 1). Theputative binding sites of SF2/ASF (Cartegni, L., and A. R. Krainer.2002. Nat. Genet. 30:377-384) and hnRNP-A1 (Kashima, T., and J. L.Manley. 2003. Nat. Genet. 34:460-463) fell within the inhibitorycis-element “Exinct”. Meanwhile, the binding site of Tra2-β1 (Hofmann,Y., et al. 2000. Proc. Natl. Acad. Sci. USA 97:9618-23) fell within thestimulatory cis-element “Conserved Tract”. Consistent with a recentreport that revealed the presence of an extended inhibitory context(termed ‘Exinct’) at the 5′ end of exon 7, in vivo selection showed thelongest tract of mutable residues at the 5′ end of exon 7 (Singh et al.,RNA 10, 1291-1305). Most importantly, in vivo selection revealed a novel7-nucleotide-long inhibitory region towards the 3′ end of exon 7, whichwas named “3′-Cluster” (Singh et al., RNA 10, 1291-1305). This clusterspans positions 45 through 51 and includes the translation terminationcodon. It also includes the last residue of a leucine codon that is notevolutionarily conserved among mammalian exon 7 (Singh et al., RNA 10,1291-1305). Deletion of leucine codon promoted exon 7 inclusion in SMN2,whereas deletion of the preceding codon had no effect, suggesting aspecific role of nucleotides that were gained (or retained) duringevolution. Another interesting feature of in vivo selection was theidentification of a long-conserved tract in the middle of exon 7.Supporting the stimulatory role of the long conserved tract, manymutations within this tract led to exon 7 exclusion in SMN1 (Singh etal., RNA 10, 1291-1305). These results suggested that the long conservedtract contains many overlapping stimulatory cis-elements, some of whichcould be tissue-specific.

Example 2 Exon 7 has a Weak 5′ Splice Site (ss)

The 5′ splice site (ss) of exon 7 is defined by sequences that arelocated at the junction of exon 7 and intron 7. One of the noteworthyoutcomes of in vivo selection of the entire exon 7 was the discovery ofthe extremely weak nature of the 5′ ss (Singh et al., RNA 10,1291-1305). It was surprising to observe the strong impact of a singlenucleotide substitution (A54G at the last position of exon 7) that notonly restored exon 7 inclusion in SMN2, but also obviated therequirement of other regulatory elements. These results showed that aweak 5′ ss of exon 7 served as the limiting factor for exon 7 inclusionin SMN2, and this weak 5′ ss thereby facilitated exon 7 exclusion inSMN2.

Example 3 Sub-Optimal U1 snRNA Base Pairing Contributes to the Weak 5′ss of Exon 7

U1 snRNA is the RNA component of the U1 snRNP, which is abundantlypresent in all cell types and plays a critical role in pre-mRNAsplicing. Loss or gain of U1 snRNA base pairing has been directly linkedto genetic disorders (Grover et al., J. Bio. Chem. 274, 15134-15143;Pagani et al., Nat. Genet. 30, 426-429; Manabe et al., Cell DeathDiffer. 10, 698-708). Having demonstrated the weak 5′ ss of exon 7 inSMN genes (Singh et al., RNA 10, 1291-1305), the potential role of poorU1 snRNA base-pairing was examined as one of the possible causes of exon7 exclusion in SMN2. To test this hypothesis, a mutant U1 snRNA wasconstructed that increased the length of the complementary region from 6to 11 nucleotides. Upon co-transfection with SMN2, this mutant U1 snRNApromoted exon 7 inclusion to the level of SMN1. The sequence-specificeffect was confirmed by co-transfection with the wild-type U1 snRNA oran empty vector, which did not promote exon 7 inclusion. Interestingly,the mutant U1 snRNA was also able to compensate for the loss of ESEassociated with Tra2. The mutant U1 snRNA also overcame the inhibitoryeffects of G1H mutations, which have been recently shown to beinhibitory (Singh et al., RNA 10, 1291-1305). Additionally, intronicmutations that extended U1 snRNA base-pairing promoted exon 7 inclusionin SMN2, further supporting the critical role of U1 snRNA base-pairing.These results confirmed the poor recruitment of U1 snRNP at the 5′ ss ofexon 7 as the major cause of exon 7 exclusion in SMN2. Multiple factorslikely contribute to poor recruitment of the U1 snRNP. Such factorsinclude the role of RNA structure, which also defines the accessibilityof the 5′ ss of the alternatively spliced exon. Role of RNA structure iswell documented in alternative splicing of exon 10 of tau, which isimplicated in Fronto-Temporal Dementia associated with Parkinsonism(Grover et al., J. Bio. Chem. 274, 15134-15143). Additional factors thatbind to intronic sequences downstream of the U1 snRNA binding site wereaccordingly predicted to play a role in affecting the accessibility ofU1 snRNP.

Example 4 Identification of an Intronic Splicing Silencer (ISS) withinIntron 7

Intronic cis-elements as the regulators of alternative splicing havebeen documented in many systems (see e.g., Lou et al., Mol. Cell. Biol.15, 7135-7142; Nishiyama et al., J. Bone Miner. Res. 18, 1716-1722). Inthe case of SMN genes, two intronic elements that modulate exon 7splicing have been identified (Miyajima, H., et al. 2002. J. Biol. Chem.277:23271-23277; Miyaso, H., et al. 2003. J. Biol. Chem.278:15825-15831). However, the trans-acting factors interacting at theseelements have not been characterized. Having confirmed the critical roleof U1 snRNA binding at the 5′ ss of exon 7, discovery of previouslyunidentified intronic elements that contribute to the suboptimal 5′ ssof exon 7 was prioritized. In initial studies to accomplish this goal,random deletions/mutations were made at different locations withinintron 7. While deletions/mutations at most locations had only marginaleffect on exon 7 inclusion in SMN2, one site was identified to showimproved exon 7 inclusion following deletion/mutation. This domain wastermed the Intronic Splicing Silencer N1 (“ISS-N1”, a sequencecomprising 5′-CCAGCAUU-3′ (SEQ ID NO: 1; refer to SEQ ID NO: 3,“5′-CCAGCAUUAUGAAAG-3′)) and was located just after the U1 snRNA bindingsite at the 5′ ss of intron 7 (FIG. 3A). To further characterize thissilencer element, a randomization-and-selection approach was adopted inwhich a small sequence stretch was first randomized to create acombinatorial library of SMN2 mutants. About 50 mutants were analyzedfor in vivo splicing activity. One mutant fully restored exon 7inclusion in SMN2, supporting the inhibitory nature of ISS-N1 (FIG. 3B,mutant ‘033’). To explore further whether an intronic cis-elementcontributed towards the weak 5′ ss, SMN2 minigene mutants with differentdeletions at the 5′ end of intron 7 were generated. The in vivo splicingpattern of these mutants was then determined, using the highlytransfectable cell line C33a (FIG. 4B). In all the deletions, the firstnine nucleotides of intron 7 were retained. These nucleotides areconserved among mammals and harbor the canonical base-pairing region forU1 snRNA, a component of U1 snRNP. First, five-nucleotide-long deletionsbetween positions 10 and 34 were made (mutants N1Δ10-14, N1Δ15-19,N1Δ20-24, N1Δ25-29 and N1Δ30-34, FIG. 4B, lanes 4-8). Analysis of thesemutants revealed two inhibitory stretches from positions 10 to 14(CCAGC) and from 20 to 24 (GAAAG), separated by a five-nucleotidesequence (AUUAU). Deletion of GAAAG produced the strongest stimulatoryeffect (mutant N1Δ20-24; FIG. 4B, lane 6), whereas deletion of CCAGCproduced a moderate but noticeable stimulatory effect (mutant N1Δ10-14;FIG. 4B, lane 4). Deletion of AUUAU produced no stimulatory effect(mutant N1Δ15-19; FIG. 4B, lane 5), indicating that this deletion likelystrengthened an inhibitory element by bringing the flanking sequencesCCAGC and GAAAG together. Indeed, when AUUAU was deleted together witheither CCAGC or GAAAG, SMN2 exon 7 inclusion increased (mutants N1Δ10-19and N1Δ15-24; FIG. 4B, lanes 9 and 10, respectively). In general, ten orfifteen nucleotide deletions that did not include CCAGC or GAAAGproduced no stimulatory effect on exon 7 inclusion (mutants N1Δ25-34 andN1Δ25-39; FIG. 4B, lanes 12 and 16, respectively).

The preceding results further revealed the ISS-N1 negative elementlocated downstream of the U1 snRNA binding site in intron 7 betweenpositions 10 and 24. To confirm that the stimulatory effect of ISS-N1deletion was not due to the creation of an enhancer element, twoadditional 20 and 25 nucleotide-long deletions were made. Both deletionsincluded the complete ISS-N1 sequence (mutants N1Δ10-29 and N1Δ10-34,FIG. 4A). Similar to ISS-N1 deletion (mutant N1Δ10-24), they restoredexon 7 inclusion in SMN2 to the level of SMN1 (FIG. 4B, lanes 17 and18). In all three mutants (N1Δ10-24, N1Δ10-29 and N1Δ10-34), the natureof the sequences downstream of the U1 snRNA binding site was different,demonstrating that exon 7 inclusion was not due to the gain of aspecific cis-element but due to the loss of ISS-N1.

To determine whether ISS-N1-mediated downregulation of exon 7 inclusionin SMN2 was tissue-specific, five additional cell lines were used thatincluded human neuroblastoma (SK-N-SH), human embryonal kidney cells(HEK 293), mouse neuroblastoma (Neuro-2a), mouse embryonalteratocarcinoma (P19) and mouse motor-neuron-like cells (NSC 34). Thesecell lines were transfected with ISS-N1-deleted SMN2 minigene (mutantN1Δ10-24 in FIG. 4A; mutant N1Δ10-24 is henceforth termed“SMN2ΔISS-N1”). Similar to the cervical carcinoma cell line C33a (FIG.4B), all cell types supported exon 7 inclusion in SMN2ΔISS-N1 (FIG. 4C).These results demonstrated that ISS-N-1-mediated suppression of SMN2exon 7 exclusion was not specific to a particular cell type.

Example 5 Selected ISS-N1 Mutants Improve Presentation of the 5′ SpliceSite of Exon 7

The predicted RNA structure at the 5′ splice site (ss) of SMN2 exon 7for wild-type, mutant 033 and mutant A54G SMN2 pre-mRNAs is shown inFIG. 6. Numbering of the sequences shown starts from exon 7. Exon 7sequence is depicted in the large-case letters. Intron 7 sequences areindicated by lower-case letters. Circled letters represent substitutionmutations. Stem-loop structure TSL2 (dark shading) was broken by A54Gsubstitution that formed co-axial stacking (light shading). Similarco-axial stacking was maintained in mutant 033 (FIG. 3B, lane 13), amutant that fully restored exon 7 inclusion. These structural resultsprovided a mechanistic rationale for targeting ISS-N1.

Example 6 Mouse Smn lacks ISS-N1

In contrast to human, the mouse genome has a single Smn gene, which isequivalent to human SMN1. Alignment of human and mouse intron 7 showedthree substitutions and three deletions in the ISS-N1 region of mouseintron 7 (FIG. 5A). There are also four additional substitutions in thefifteen-nucleotide-long stretch following ISS-N1. To test whetherdifferences between human and mouse intronic sequences affected splicingof exon 7, the ISS-N1 region of the SMN2 minigene was altered to mouseSMN sequence at the corresponding residues by introducing threesubstitutions and three deletions (mutant SMN2/MS8, FIG. 5A). This SMN2mutant fully restored exon 7 inclusion (FIG. 5B, lane 11). To furtherdissect the role of acquired mutations within ISS-N1, the splicingpattern of SMN2 mutants that incorporated different combinations ofdeletions and substitutions corresponding to mouse sequences was tested.Consistent with the inhibitory role of acquired mutations, a single A18Usubstitution in the middle of ISS-N1 increased exon 7 inclusion in SMN2from ˜19% to ˜34% (mutant SMN2/MS1, FIG. 5B, lane 4). When A18U wascombined with G20A, the exon 7 inclusion increased to ˜50% (mutantSMN2/MS4, FIG. 5B, lane 7). A deletion of the three nucleotides frompositions 11 to 13 accounted for an increase of ˜28% (from 19% to 47%;mutant SMN2/MS6, FIG. 5B, lane 9). When this triple deletion wascombined with an adjacent C10U substitution, exon 7 inclusion increasedto ˜66% (mutant SMN2/MS7, FIG. 5B, lane 10). As a control, substitutionswithin sequences downstream of ISS-N1 were also made. Contrary tomutations within ISS-N1, the effects of these substitutions on SMN2splicing were negligible (mutants SMN2/MS3 and SMN2/MSS, FIG. 5B, lanes6 and 8, respectively). These results were fully consistent with thedeletion mutant results that defined the approximate boundary of ISS-N1within human intron 7 (FIGS. 4A and 4B).

Example 7 Antisense RNA-oligo targeting ISS-N1 promotes exon 7 inclusionin SMN2

Antisense oligonucleotides have traditionally been used to block genefunction, e.g., a DNA oligonucleotide anneals to mRNA and mRNA isdegraded by an RNase H response (Sazani and Kole, J. Clin. Invest. 112,481-486). On the contrary, antisense RNA oligonucleotides do not elicitRNase H responses and offer an entirely new set of possibilities inwhich they could be used to modulate genes without degrading mRNA orpre-mRNA (precursor mRNA, e.g., nuclear, unspliced transcripts; Sazaniand Kole, J. Clin. Invest. 112, 481-486; Kole, Oligonucleotides 14,65-74; Mercatante et al., Curr. Cancer Drug Targets 1, 211-30). RNAantisense oligonucleotides could also be used to test/confirm thepresence or absence of a cis-element associated with protein and/or RNAstructure. Having identified such an element (ISS-N1) in SMN2 intron 7,it was desired to confirm the inhibitory nature of this element, throughuse of an antisense oligonucleotide-based approach. A 20 nt long RNAoligonucleotide was synthesized that anneals to ISS-N1 (termed“anti-N1,” 5′-AUUCACUUUCAUAAUGCUGG-3′, SEQ ID NO: 39, FIG. 7A). Thisoligonucleotide was 2′-O-methyl modified and additionally possessed aphosphorothioate backbone for greater stability in vivo (refer toMaterials and Methods). Such oligonucleotides provide greater antisenseeffect through enhanced annealing. Modified oligonucleotides are alsoresistant to ribonucleases (enzymes that degrade RNAs), which areabundantly present in the cell. Different concentrations of antisenseoligonucleotides in combination with a fixed concentration of an SMN2minigene were used to transfect C33a cells (these cells show hightransfection efficiency). Lipofectamine™ 2000 reagent (Invitrogen) wasused for co-transfection of anti-N1 with the SMN2 minigene. Splicedproducts were analyzed after 20 hours of transfection. The anti-N1oligonucleotide produced a dramatic stimulatory effect on inclusion ofSMN2 exon 7 in the SMN2 splice product, thereby confirming theinhibitory nature of the ISS-N1 domain that is the target of the anti-N1oligonucleotide. The stimulatory effect was pronounced even at thelowest concentrations of anti-N1 oligonucleotide (25 nM). Controloligonucleotides that did not anneal to the ISS-N1 site showed nostimulatory effect (FIG. 7B). The effect of anti-N1 oligonucleotidetreatment was also observed in a longer SMN2 minigene that containedgenomic sequence from exons 4-to-8 (data not shown). These resultsconfirmed the sequence-specific effect of anti-N1 oligonucleotidetreatment.

Example 8 The Anti-N1 Oligonucleotide Effect was Specific to the ISS-N1Target

The specificity of the antisense anti-N1 oligonucleotide was initiallydetermined by use of four overlapping antisense RNA-oligonucleotides(anti-N1 and three additional oligonucleotides termed anti-N1+10,anti-N1+20 and anti-N1+30, respectively; FIG. 8A). These antisenseoligonucleotides were complementary to intronic sequences downstream ofU1 snRNA binding site, including ISS-N1 region were generated. Antisenseoligonucleotide Anti-N1 fully blocked ISS-N1 by annealing to a20-nucleotide-long sequence starting from position 10 of intron 7 (FIG.8A). Antisense oligonucleotide Anti-N1+10 partially targeted ISS-N1 byannealing to a sequence starting from position 20 of intron 7 (FIG. 8A).Anti-N1+20 and Anti-N1+30 annealed to sequences downstream of ISS-N1starting from positions 30 and 40, respectively (FIG. 8A). To increasethe intracellular stability of these oligonucleotides, each weremodified to comprise a phosphorothioate backbone and 2′-O-methylmodification in the sugar moiety. Antisense oligonucleotides withsimilar modifications have previously been used to correct aberrantsplicing in vivo (Crooke, S. T. 2004. Curr. Mol. Med. 4:465-487; Lu Q.L., et al. 2005. Proc. Natl. Acad. Sci. USA 102:198-203).

The effect of antisense oligonucleotides on SMN2 splicing was determinedby co-transfection of C33a cells with 1.0 μg of SMN2 minigene and 50 nMof antisense oligonucleotides. Consistent with the result obtained fordeletion mutant N1Δ10-29 above (FIG. 4B), the Anti-N1 oligonucleotiderestored exon 7 inclusion in SMN2 to the level of SMN1 (FIG. 8B, lane4). The complete restoration of SMN2 exon 7 inclusion was also observedat Anti-N1 concentration as low as 10 nM when the amount of SMN2minigene was reduced to 0.1 μg (not shown). The three antisenseoligonucleotides tested that annealed to intron 7 sequences downstreamof ISS-N1 did not produce any stimulatory effect (FIG. 8B, lanes 5-7).The described results were reproducible with two different batches ofantisense oligonucleotides synthesized at different times. These resultsconfirmed the sequence-specific effect of antisense oligonucleotidetreatment and validated ISS-N1 as the target for antisense-mediatedtherapy. The splicing effect of anti-ISS-N1 oligonucleotide wasadditionally observed at all concentrations tested (data not shown).

To confirm that Anti-N1 did not globally impact alternative splicing ofother exons, C33a cells were transfected with different minigenes in thepresence and absence of Anti-N1, and in vivo splicing patterns weredetermined. These minigenes were randomly selected and represented arobust mix of exon-including and exon-excluding cassettes (FIG. 8C).Among minigenes that showed low skipping of exons were pTBEx9-V456F(−25% skipping of CFTR exon 9), pTBEx12-50A (˜20% skipping of CFTR exon12), pTBApo-ISE3m (˜15% skipping apoA-II exon 3), Casp3 (˜6% skipping ofCaspase 3 exon 6) and CMV-Fas (wt; ˜6% skipping of Fas exon 6). Amongminigenes that showed mostly skipping of exons were SI9/LI10 (˜85%skipping of Tau exon 10) and CMV-Fas (U-20C; ˜98% skipping of Fas exon6). In the above experiments, 50 nM of Anti-N1 was used, and theminigene-containing plasmid concentration was decreased to 0.1 μg. Ahigh Anti-N1 to minigene ratio was deliberately chosen to detect anynon-specific effect of the Anti-N1 oligonucleotide at a non-limitingAnti-N1 concentration. No appreciable change in the splicing pattern ofany of the minigenes co-transfected with Anti-N1 was observed (FIG. 8C),demonstrating the specificity of the Anti-N1 splicing effect to SMNintron 7.

Example 9 Stimulatory Effect of Anti-N1 was Caused by Base Pairing withthe Target Sequence

To prove that the stimulatory effect of Anti-N1 was solely due to theblocking of ISS-N1, co-transfection experiments were performed with SMN2minigenes that had random mutations within ISS-N1 (FIG. 9A). Of note,from a large library of ISS-N1 mutants, only those containingsubstitutions that did not effect SMN2 exon 7 inclusion but thatabrogated base pairing between ISS-N1 and Anti-N1 were chosen fortesting. Consequently, upon co-transfection, Anti-N1 did not stimulateexon 7 inclusion in any of these mutants (FIG. 9C). Remarkably, thestimulatory effect of Anti-N1 completely disappeared even with a mutantthat had only a two base-pair mismatch with Anti-N1 (FIG. 9C, lanes 7,8). Notably, Anti-N1 also reduced SMN1 exon 7 exclusion from ˜5% to ˜1%(FIG. 9C, compare lane 1 with lane 2), suggesting that blocking ofISS-N1 had the potential to upregulate SMN expression from both SMN1 andSMN2. To further confirm that the observedantisense-oligonucleotide-mediated stimulatory effect was due to basepairing between the antisense oligonucleotide and ISS-N1, a mutantoligonucleotide was included in these co-transfection experiments(Anti-I7-25, FIGS. 9B and C). This oligonucleotide formed perfect basepairing with the ISS-N1 region of SMN2/17-25 minigene. The SMN2/17-25construct was chosen for targeting because its ISS-N1 region containsfive substitutions (33% change that includes more than two-fold increasein U residues and 33% decrease in G residues in a 15-nucleotide longISS-N1). Therefore, by using Anti-I7-25, the effect of base pairingcould be tested in a mutant that retained inhibitory function despite amajor change in the sequence composition. Remarkably, Anti-I7-25produced about a six-fold increase in exon 7 inclusion in SMN2/17-25(FIG. 9C, lane 15). At the same time, Anti-I7-25 had no stimulatoryeffect on the splicing pattern of SMN1 or SMN2 minigenes. Similarly,Anti-I7-25 did not effect the splicing pattern of mutants SMN2/I7-08 andSMN2/I7-09. The above results conclusively confirmed the inhibitory roleof ISS-N1 and demonstrated that the antisense oligonucleotides produceda specific stimulatory effect on exon 7 inclusion when ISS-N1 wasblocked by base pairing.

Example 10 Deletion of ISS-N1 Rescued Exon 7 Inclusion in Mutants withAbrogated Positive cis-Elements

To assess the relative impact of ISS-N1 on exon 7 splicing, SMN1 mutantswere generated in which ISS-N1 deletion was combined with the abrogationof one of the stimulatory cis-elements. In particular, the impact ofISS-N1 deletion on inhibitory substitutions within four positivecis-elements, i.e., intronic element 2 (Miyajima, H., et al. 2002. J.Biol. Chem. 277:23271-23277; Miyaso, H., et al. 2003. J. Biol. Chem.278:15825-15831), Tra2-ESE (Hofmann, Y., et al. 2000. Proc. Natl. Acad.Sci. USA 97:9618-23), conserved tract (Singh, N. N., et al. 2004. RNA10:1291-1305) and a critical guanosine residue at the first position ofexon 7 (Singh, N. N., et al. 2004. Biochem. Biophys. Res. Commun.315:381-388; Singh, N. N., et al. 2004. RNA 10:1291-1305), was tested.The diagrammatic representation of the relative positioning of theseelements is shown in FIG. 10A. Demonstrating the cross-exon effect,deletion of ISS-N1 fully restored exon 7 inclusion in SMN1 harboringguanosine-to-uridine substitution at the first position of exon 7(mutant SMN1ΔISS-N1/1U, FIG. 10B, lane 9). Similarly, deletion of ISS-N1promoted exon 7 inclusion in SMN1 mutants with abrogated element 2(mutant SMN1ΔISS-N1/Abr-E2, FIG. 10B, lane 8) and abrogated Tra2-ESE(mutant SMN1ΔISS-N1/Abr-Tra2, FIG. 10B, lane 10). Although lessprominent, the stimulatory effect of ISS-N1 deletion was clearlydetectable in an SMN1 mutant with an abrogated conserved tract (mutantSMN1ΔISS-N1/Abr-CT, FIG. 10B, lane 11). These results demonstrated forthe first time that the single, intronic ISS-N1 element had a profoundimpact on exon 7 splicing in SMN genes.

Example 11 ISS-N1 Showed Portability within SMN2 Intron 7 as well as ina Heterologous System

To demonstrate the portability of ISS-N1 within intron 7, four mutantsin which ISS-N1 was moved away from the 5′ ss of SMN2 exon 7 weregenerated (FIG. 11A, upper panel). The splicing patterns of thesemutants were then tested. While moving ISS-N1 five nucleotides away fromits original position fully retained the inhibitory impact of ISS-N1(FIG. 11B, lane 3), there was a slight decrease in the inhibitory effect(from ˜89% to ˜72%) when ISS-N1 was displaced an additional fivenucleotides (FIG. 11B, lane 4). Moving of ISS-N1 twenty nucleotides awayfrom its original position produced a dramatic effect in which exon 7exclusion decreased from ˜89% to ˜18% (FIG. 11B, lane 5). Furtherdisplacement of ISS-N1 by another fifteen nucleotides completelyeliminated the inhibitory impact of ISS-N1 and restored exon 7 inclusionin SMN2 to the level of SMN1 (FIG. 11B, lane 6). These resultsdemonstrated the limited portability of ISS-N1, as the inhibitory effectof ISS-N1 required close proximity to the 5′ ss.

The observation that moving ISS-N1 five nucleotides away from itsoriginal position fully retained the inhibitory impact of ISS-N1 (mutantISS-N-1-M5 in FIG. 11A), suggested that the nature of thefive-nucleotide-long sequence UGAAU that immediately preceded ISS-N1 wasunable to break the inhibitory context responsible for theISS-N-1-mediated exclusion of exon 7. To determine whetherISS-N1-associated inhibitory impact was context-specific, the effects offive-nucleotide-long insertions immediately preceding ISS-N1 wereexamine (FIG. 11A, lower panel). Insertion of five guanosine residuesthat created an eight-nucleotide-long GC-rich stretch upstream of ISS-N1did not change exon 7 splicing in SMN2 (FIG. 11B, lane 12). Similarly,insertion of five adenosines upstream of ISS-N1 had negligible effect onsplicing pattern of SMN2 (FIG. 11B, lane 9). Insertion of five Cresidues created a stretch of seven cytosines and partially improvedexon 7 inclusion in SMN2 (FIG. 11B, lane 11). However, insertion of fiveuridines alleviated the inhibitory effect of ISS-N1 and substantiallyincreased SMN2 exon 7 inclusion (FIG. 11B, lane 10). These resultsshowed that the inhibitory impact of ISS-N1 element was not onlydependent upon proximity of ISS-N1 to the 5′ ss, but was also influencedby the nature of sequences immediately upstream of ISS-N1. As controls,the impacts of identical insertion mutations in SMN1 were tested. Theseinsertions possessed no independent inhibitory effects, as none of thesemutations effected SMN1 exon 7 splicing (FIG. 11B, lanes 13-17).

After the limited portability of ISS-N1 within intron 7 of SMN2 wasdemonstrated, the portability of ISS-N1 in a heterologous context wasexamined, using a Casp3 minigene that contains Caspase 3 genomicsequence from exon 5 through exon 7. This minigene recapitulated partialskipping of the endogenous Caspase 3 exon 6, as initially reported byHuang et al (Huang, Y., et al. 2001. Biochem. Biophys. Res. Commun.283:762-769). Insertion of an AvrII restriction site downstream of exon6 did not change the splicing pattern of exon 6 (compare lane 1 in FIG.11D with lane 13 in FIG. 8C) but enabled insertion of ISS-N1, allowingfor testing of the portability of this element in a heterologouscontext. As shown in FIG. 11C, ISS-N1 sequence was inserted ninenucleotides away from the 5′ ss of exon 6 (mutant Casp3ISS-N1, FIG.11C). This position was selected because ISS-N1 was located at theidentical position within intron 7 of SMN genes (FIG. 4A).Interestingly, ISS-N1 insertion caused about a six-fold increase inskipping of Casp3 exon 6 (FIG. 11D, compare lane 1 with lane 3).Blocking of ISS-N1 by a fifteen-nucleotide-long antisense oligo(Anti-ISS-N1/15) fully restored the inclusion of Casp3 exon 6 (FIG. 11D,compare lane 4 with lane 3). The SMN2 minigene was used as the positivecontrol for testing the effect of Anti-ISS-N1/15. As expected,Anti-ISS-N1/15 fully restored exon 7 inclusion in transcripts derivedfrom the SMN2 minigene. The above results confirmed that ISS-N1 was aportable inhibitory element even in a heterologous context. Remarkably,ISS-N1 was able to exert its inhibitory impact despite the presence of Gresidues at the first and the last position of skipping exon 6 in Casp3(FIG. 11C). Previously, it has been shown that the presence of a Gresidue at the last position of exon 7 in SMN2 renders exon 7 exclusionundetectable even in a sensitive radioactive assay (Singh, N. N., et al.2004. RNA 10:1291-1305).

Example 12 Anti-N1 Oligonucleotide Treatment was Effective in SMA type IFibroblasts

The effect of antisense anti-N1 oligonucleotide treatment in SMA patientcells was examined. Patient-derived SMA type I fibroblasts(specifically, the SMA type I fibroblast line, GM03813, that carries theentire SMN2 gene but no SMN1 gene) were transfected (usingLipofectamine™ 2000 (Invitrogen)) in parallel with antisenseoligonucleotides anti-N1, anti-N1+10, anti-N1+20, and anti-N1+30. Due tothe absence of SMN1 in GM03813 cells, these cells produced high levelsof exon 7-excluded products (FIG. 12A, lane 2). As expected,transfection of GM03813 cells (SMA cells) with increasing concentrationsof Anti-N1 yielded increased exon 7 inclusion (not shown). The minimumconcentration at which the level of exon 7 inclusion in SMA cellsincreased to the level observed in normal fibroblasts was 5 nM.Consequently, SMA cells were transfected with 5 nM of antisenseoligonucleotides (Anti-N1, Anti-N1+10, Anti-N1+20 and Anti-N1+30). Theannealing positions of the preceding oligonucleotides were the same asshown in FIG. 8A. Only Anti-N1 restored exon 7 inclusion in SMA cells toa level comparable to that observed for normal fibroblasts (FIG. 12A).No splicing effect was observed for oligonucleotides anti-N1+10,anti-N1+20, and anti-N1+30 (FIG. 12A). The experiments were reproduciblewith two batches of oligonucleotides. These results showed that theISS-N1 target site is accessible in the endogenous SMN2 transcript,which is several-fold bigger than the transcript derived from the SMN2minigene used in previous examples. Most significantly, these resultsalso confirmed that the anti-N1 oligonucleotide was specific and highlyeffective against the ISS-N1 target. The fact that anti-N1oligonucleotide was highly effective at low concentrations indicatesthat non-specific effects will be decreased upon therapeuticadministration and also makes the therapeutic easier to deliver to apatient (e.g., delivery may be made to a patient with efficacymaintained even if only low concentrations reach the target cells of thepatient, e.g., the patient's neuronal cells). Thus, the precedingresults validated the inhibitory role of ISS-N1 in the context of theendogenous SMN2 transcript.

To confirm that Anti-N1 did not cause aberrant splicing of other exonsin patient cells, the following experiment was performed. RNApreparations from untreated fibroblasts and fibroblasts treated with 5nM of Anti N1 (FIG. 12A, lanes 2 and 3) were used to determine thesplicing pattern of a limited number of the randomly selected endogenousgenes that are known to generate alternatively spliced products (Chen,X., et al. 2003. Cell Biol. Int. 27:491-496; Huang, Y., et al. 2001.Biochem. Biophys. Res. Commun. 283:762-769; Mahotka, C., et al. 1999.Cancer Res. 59:6097-6102; Mercatante, D. R., et al. 2002. J. Biol. Chem.277:49374-49382; Park, V. M., et al. 1998. Hum. Genet. 103:382-385). Nodetectable change was observed in the splicing pattern of any of theexamined genes, including Tra2, which produces Tra2-β1 (compare 868base-pair band in lanes 9 and 10 in FIG. 12B; Chen, X., et al. 2003.Cell Biol. Int. 27:491-496). Tra2-β1 plays a stimulatory role in SMN2exon 7 inclusion, but is downregulated in SMA patient cells (Helmken,C., et al. 2003. Hum. Genet. 114:11-21). Also, the splicing pattern ofSMN exons 3 and 5 remained unaffected in patient cells treated withAnti-N1. These exons were shown to undergo alternative splicing(Hsieh-Li, H. M., et al. 2000. Nat. Genet. 24:66-70). Despite the smallsample size, these results did not support a global effect of Anti-N1 onthe alternative splicing of other genes.

Example 13 Anti-N1 Oligonucleotide-Treated Fibroblasts ExpressedFull-Length SMN Protein

Anti-N1-treated fibroblasts were examined for production of SMN protein.Antisense oligonucleotide (anti-N1) treatment was demonstrated torestore levels of SMN protein in SMA patient cells in the following twoexperiments (FIGS. 12C and D). In the first, SMA fibroblasts weretransfected with anti-N1 oligonucleotide and cell lysates were prepared48 and 72 hours post-transfection. Levels of SMN protein increased incells treated with anti-N1 as compared to untransfected cells (FIG.12C). To ensure even protein loading, membranes were stained with SyproRuby Protein Blot stain (Bio-Rad). In addition, SMN protein levels werecompared to alpha-tubulin levels as an internal control (though elevatedalpha-tubulin levels have consistently been observed in patientfibroblasts as compared to normal fibroblasts). In the secondexperiment, SMA cells were treated with 5 or 15 nM of Anti-N1. Theseconcentrations fell within the lower range of Anti-N1 concentrationsthat corrected SMN2 splicing in patient cells. Both concentrationscaused a significant increase in SMN protein level (FIG. 12D). Thesewere the first reported results in which anantisense-oligonucleotide-assisted increase in the level of SMN proteinin patient cells was detected by western blot. This almost certainlyoccurred due to a more than five-fold increase in exon 7 inclusion fromthe endogenous SMN2 gene (FIG. 12A). The level of SMN protein remainedelevated for five days after transfection. To confirm the specificity ofAnti-N1-induced stimulation, a scrambled oligonucleotide was used as anegative control. As shown in FIG. 12D (lanes 2 and 4), thisoligonucleotide did not produce any detectable increase in the levels ofSMN protein. These results confirmed that antisense targeting of theISS-N1 site not only caused inclusion of exon 7 in SMN2 mRNA, but alsothat elevated levels of exon 7-containing SMN2 mRNA directly led toelevated levels of SMN protein in SMA patient cells.

Example 14 Variant Forms of ISS-N1 Sequence Possessing Splice SiteInhibitory Activity

The following variant forms of ISS-N1 seqeunce were tested in the assaydescribed in Example 4:

(SEQ ID NO: 3) Sequence# N-IN7-0WT: CCAGCAUUAUGAAAG (SEQ ID NO: 40)Sequence# N-IN7-001: CUAGCAACAUGAAAG (SEQ ID NO: 41) Sequence#N-IN7-002: ACAGGCCGAUGAAAG (SEQ ID NO: 42) Sequence# N-IN7-003:UGAGAACCAUGAAAG (SEQ ID NO: 43) Sequence# N-IN7-006: CGAGUUAGAUGAAAG(SEQ ID NO: 44) Sequence# N-IN7-007: CCAGGGGAAUGAAAG (SEQ ID NO: 45)Sequence# N-IN7-008: CCAGAAGGAUGAAAG (SEQ ID NO: 46) Sequence#N-IN7-009: CGAGUCUCAUGAAAG (SEQ ID NO: 47) Sequence# N-IN7-01A:CGAGCGGUAUGAAAG (SEQ ID NO: 48) Sequence# N-IN7-02A: GGAGCGGUAUGAAAG(SEQ ID NO: 49) Sequence# N-IN7-20A: CCAGAGGUAUGAAAG (SEQ ID NO: 50)Sequence# N-IN7-21A: CCAGCGGUAUGAAAG (SEQ ID NO: 51) Sequence#N-IN7-22A: CCAGCAGUAUGAAAG (SEQ ID NO: 52) Sequence# N-IN7-008:CCAGAAGGAUGAAAG (SEQ ID NO: 53) Sequence# N-IN7-011: UAAGCCCUAUGAAAG(SEQ ID NO: 54) Sequence# N-IN7-020: CUAGUUUUAUGAAAG (SEQ ID NO: 55)Sequence# N-IN7-025: CCUUAAUUUAGAAAG (SEQ ID NO: 56) Sequence#N-IN7-R1-AA: AAAGCAUUAUGAAAG (SEQ ID NO: 57) Sequence# N-IN7-R1-UU:UUAGCAUUAUGAAAG (SEQ ID NO: 58) Sequence# N-IN7-R1-GU: CGUGCAUUAUGAAAG(SEQ ID NO: 59) Sequence# N-IN7-R3006: CUGUCAUUAUGAAAG (SEQ ID NO: 60)Sequence# N-IN7-R3C10: CUUUCAUUAUGAAAG (SEQ ID NO: 61) Sequence#N-IN7-R3C11: CAUUCAUUAUGAAAG (SEQ ID NO: 62) Sequence# N-IN7-R3B17:CCAGCAUUAUGAUUA (SEQ ID NO: 63) Sequence# N-IN7-R3A21: CCAGCAUUAUCUAAG(SEQ ID NO: 64) Sequence# N-IN7-R3C17: CCAGCAUUAUCCCAG (SEQ ID NO: 65)Sequence# N-IN7-R3A07: CCAGCAUUAUUUUAG (SEQ ID NO: 66) Sequence#N-IN7-R0709: CCAGCAUUAAUCAGGWithin the above sequences, underlined nucleotides indicate mutations inthe ISS-N1 wild-type sequence. All variant forms above retained theinhibitory function of ISS-N1.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of increasing the level of exon 7-containing SMN2 mRNA in acell comprising contacting the cell with an oligonucleotide, whicholigonucleotide comprises a sequence: at least 80% complementary tointron 7 of the SMN2 gene over the entire length of the oligonucleotideand at least 85% complementary to the sequence set forth in SEQ ID NO:1or SEQ ID NO:3; such that the level of exon 7-containing SMN2 mRNA inthe cell is increased.
 2. The method of claim 1, wherein theoligonucleotide is 15-40 nucleotides in length and is 100% complementaryto intron 7 of the SMN2 gene over the full length of theoligonucleotide.
 3. The method of claim 1, wherein the oligonucleotideis complementary to the sequence set forth in SEQ ID NO:1.
 4. The methodof claim 1, wherein the oligonucleotide is complementary to the sequenceset forth in SEQ ID NO:3.
 5. The method of claim 1, wherein theoligonucleotide is 15-40 nucleotides in length.
 6. The method of claim1, wherein the oligonucleotide is about 10-15 nucleotides in length. 7.The method of claim 1, wherein the oligonucleotide is about 15-20nucleotides in length.
 8. The method of claim 1, wherein theoligonucleotide comprises at least one modified nucleotide.
 9. Themethod of claim 1, wherein the oligonucleotide comprises at least onemodified sugar moiety.
 10. The method of claim 1, wherein theoligonucleotide comprises at least one morpholino moiety.
 11. The methodof claim 1, wherein the oligonucleotide comprises at least one 2′-deoxyribonucleotide.
 12. The method of claim 1, wherein the 2′-deoxyribonucleotide is 2′-deoxy adenosine or 2′-deoxy guanosine.
 13. Themethod of claim 1, wherein the oligonucleotide comprises at least onemodified nucleotide comprising a modified sugar moiety which is modifiedat the 2′-position.
 14. The method of claim 13, wherein the modifiedsugar moiety comprises a 2-substituent selected from the groupconsisting of: H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, and ON, where R isa C₁-C₆ alkyl, alkenyl, or alkynyl and halo is F, Cl, Br or I.
 15. Themethod of claim 1, wherein the oligonucleotide comprises at least onemodified nucleotide selected from the group consisting of2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine and2′-amino-butyryl-pyrene-uridine.
 16. The method of claim 1, wherein theoligonucleotide comprises at least one modified nucleotide selected fromthe group consisting of 5-bromo-uridine, 5-iodo-uridine,5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 5-fluoro-cytidine, and5-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine, and5-amino-allyl-uridine.
 17. The method of claim 1, wherein theoligonucleotide comprises at least one modified linkage.
 18. The methodof claim 17, wherein the at least one modified linkage is aphosphorothioate linkage.
 19. The method of claim 1, the oligonucleotidecomprises at least one locked nucleic acid (LNA) nucleotide.
 20. Amethod of increasing the level of exon 7-containing SMN2 mRNA in anorganism, comprising administering to the organism an oligonucleotide,which oligonucleotide comprises a sequence: at least 80% complementaryto intron 7 of the SMN2 gene over the entire length of theoligonucleotide and at least 85% complementary to the sequence set forthin SEQ ID NO:1 or SEQ ID NO:3, such that the level of exon 7-containingSMN2 mRNA in the organism is increased.
 21. The method of claim 20,wherein the organism is a mammal.
 22. The method of claim 20, whereinthe organism is a human.
 23. The method of claim 20, wherein the humanhas spinal muscular atrophy (SMA).
 24. A method of treating spinalmuscular atrophy (SMA) in a patient, comprising administering to thepatient an oligonucleotide, which oligonucleotide comprises a sequence:at least 80% complementary to intron 7 of the SMN2 gene over the entirelength of the oligonucleotide and at least 85% complementary to thesequence set forth in SEQ ID NO:1 or SEQ ID NO:3; in a dose effective toincrease the level of exon 7-containing SMN2 mRNA in cells of thepatient, such that SMA in the patient is treated.
 25. A method forinhibiting an SMN2 pre-mRNA intronic splicing silencer site in a cell orcell extract comprising contacting the cell with an oligonucleotide 100%complementary to the ISSN-N1 sequence set forth in SEQ ID NO:1, suchthat the SMN2 intronic splicing silencer site is inhibited.
 26. A methodfor inhibiting an SMN2 pre-mRNA intronic splicing silencer site in anorganism comprising administering to the organism an oligonucleotide100% complementary to the sequence set forth in SEQ ID NO:1, such thatthe SMN2 intronic splicing silencer site is inhibited.
 27. A method ofadministering an oligonucleotide to a subject comprising comprisingadministering to a subject an oligonucleotide, which oligonucleotidecomprises a sequence: at least 80% complementary to intron 7 of the SMN2gene over the entire length of the oligonucleotide and at least 85%complementary to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3;wherein the oligonucleotide is administered at a dose effective toincrease the level of exon 7-containing SMN2 mRNA in cells of thesubject.
 28. The method of claim 27, wherein the subject is sufferingfrom amyotrophic lateral sclerosis (ALS).
 29. The method of claim 1,wherein the method is performed in vivo.
 30. The method of claim 1,wherein the method is performed in vitro.
 31. The method of claim 9,wherein the modified sugar moiety comprises a 2′OCH₃.
 32. The method ofclaim 17, wherein each linkage of the oligonucleotide is aphosphorothioate linkage.
 33. The method of claim 1, wherein theoligonucleotide comprises at least one bicyclic nucleotide.
 34. Themethod of claim 1, wherein each nucleotide of the oligonucleotide is amodified nucleotide.
 35. The method of claim 1, wherein each nucleotideof the oligonucleotide comprises a modified sugar moiety.
 36. The methodof claim 1, wherein each nucleotide of the oligonucleotide is a modifiednucleotide and each modified nucleotide comprises the same modification.37. The method of claim 36, wherein each nucleotide of theoligonucleotide comprises a bicyclic nucleotide.
 38. The method of claim36, wherein each nucleotide of the oligonucleotide comprises a modifiedsugar moiety which is modified at the 2′-position.
 39. The method ofclaim 38, wherein the modified sugar moiety comprises a 2-substituentselected from the group consisting of: H, OR, R, halo, SH, SR, NH₂, NHR,NR₂, and ON, where R is a C₁-C₆ alkyl, alkenyl, or alkynyl and halo isF, Cl, Br or I.
 40. The method of claim 38, wherein each modified sugarmoiety comprises a 2′OCH₃.
 41. The method of claim 28, wherein thesubject is suffering from spinal muscular atrophy (SMA).
 42. The methodof claim 30, wherein the cell is selected from the group consisting of aspinal muscular atrophy (SMA) patient-derived neuronal cell, a spinalmuscular atrophy (SMA) patient-derived muscle cell or a spinal muscularatrophy (SMA) patient-derived fibroblast.
 43. The method of claim 30,wherein the cell is selected from the group consisting of an embryonicstem cell, an embryonic stem cell extract, a neuronal stem cell and aneuronal stem cell extract.